Darksiders II takes place parallel to the previous game. In the prologue, it is revealed that the Four Horsemen (War, Strife, Fury, and Death) are the last of the Nephlim, fusions of angels and demons who waged a bloody war on the rest of creation. In order to preserve the balance of the Universe, the Four, who had grown tired of the conquest, received incredible powers from the Charred Council in exchange for slaughtering the rest of the Nephilim. The Horseman Death trapped the souls of his fallen brethren in an amulet, earning the title of Kinslayer, among others (though he kept its preservation a secret, since the Council ordered the Nephilims' souls destroyed).

While War is charged with his crimes, the horseman Death, sure that his brother is innocent, sets out on a personal mission to erase his brother's 'crime' and resurrect humanity. He first travels to the Icy Veil—a dimension between the three kingdoms of Heaven, Hell, and Earth—to seek the Keeper of Secrets for proof of War's innocence and the way to restore Earth. The Keeper of Secrets, also known as the Crowfather, tells Death that he must go to the Tree Of Life in order to restore humanity. The Crowfather, bitter about being given the Nephilim amulet (which torments him without end), refuses to let Death pass and attacks him in the shape of War. Death kills Crowfather, but the Amulet shatters and embeds itself in his chest, knocking him out and sending him into a portal created by the Crowfather's death.

Death wakes up in the Forge Lands, a world populated by the Makers—physically imposing beings who are the creators of all worlds. He learns that their world, and many others, has been overrun with Corruption, a dark force that has blocked off the Tree of Life and has taken over many of the Maker's constructs. The Makers had crafted a massive Guardian to combat the Corruption, but had to abandon it before finishing the task due to the surrounding threats. With the help of constructs and a Maker named Karn, Death eventually reaches the Guardian, but upon activation, the Guardian is tainted by Corruption and goes on a rampage. Death battles the Guardian and destroys it, allowing it to be reassembled without Corruption. The Guardian self-destructs in the grasp of the creature blocking the path to the Tree of Life, which allows Death to reach his goal. Upon reaching the entrance, however, Death is seized by Corruption and is dragged into the Tree.

Death is transported to the Land of the Dead, where he encounters the merchant Ostegoth. From the Merchant, he learns that in order to resurrect humanity, Death must find the Well of Souls, and to that end, he must speak with the Bone King. After completing an array of trials to gain an audience with the Bone King, Death is transported to the City of the Dead to find a soul capable of telling Death what he needs to know about the Well. After facing off against a creature housing all of mankind's souls, Death meets the soul the Bone King alluded to: the Crowfather's. The Crowfather tells Death that while the souls of humanity are no longer bound in a host, they have merely been transported to the Well. Death learns that the Well of Souls has power over life and death, and that with it, the spirits of all things living are cleansed and renewed before they are sent out to be reborn. In order to access it, Death would need two keys: one kept by the angels and one kept by the demons. Death asks why he and the other riders never knew of such things, to which the Crowfather replies that power must be tempered with ignorance and that the Council feared what the Horsemen would do if they ever learned the truth about the Well's power, that they might try to resurrect their kin.

Death first seeks out the key held by the angels and is sent to an outpost of Heaven called Lostlight, which is being assaulted by Corruption. At the Ivory Tower, he meets the Archon, who tells Death that his answers are in the Ivory Citadel, which is shrouded in darkness and Corruption. The Archon sends Death to Earth to bring back the Rod of Arafel, a powerful holy weapon, so that the way to the Ivory Citadel could be cleared. On Earth, Death encounters remnants of the Hellguard being led by Uriel. With their help, Death reassembles the shattered rod. Upon returning to Lostlight, Death eventually realizes that the Archon has possessed the key the entire time and that the angel has fallen to Corruption himself. Death confronts the Archon, kills him, and acquires the first key. Death then proceeds to Shadow's Edge, described as a dark reflection of Lostlight. When Death arrives, he sees that the world itself is in the process of being devoured by Corruption. Intent on acquiring the second key, Death travels to the fortress home of the demon lord, Samuel, but finds Lilith , a female demon-queen that created the Nephilim (she thus refers to herself as Death's mother, which he angrily denies). Death learns through Lilith that Samael is gone, that Death will be able to meet with the demon lord by utilizing a time portal. Before he leaves, Lilith urges Death to "follow [his] heart" and revive his brethren when he finds the Well of Souls. After testing Death in a fierce battle, Samael gives Death the demon key, saying that no matter what happens, it should prove to be an interesting show. With both keys in hand, Death returns to the Tree of Life and has his penultimate meeting with the Crowfather, who reminds him of what's at stake: the fate of two races, humanity and the Nephilim. He warns that Corruption has chosen a named champion to block Death's efforts.

Death enters using the keys and proceeds to the Tree's core where he is met by Absalom. The Nephilim taunts Death by saying that the reason he is not harmed by Corruption is that the horseman is already "blackened by the sin of betrayal." Absalom and Death battle, and Death defeats Absalom just as he did eons ago. It is then that the Crowfather appears one last time and explains that Death may tap the Well's power to restore one race, but a sacrifice is needed. Warned that choosing one race will forever doom the other, Death chooses to save his brother War and sacrifices the souls of the Nephilim, still trapped in the scar on his chest, for humanity by leaping into the Well. An epilogue retells the final moments from the first game, with Uriel questioning if War intends to wage his campaign against the Charred Council alone, to which War replies "No, not alone." The scene then shows the arrival of the other Horsemen. The narrator states "And the number of the Horsemen shall ever be four,", and the Horsemen appear: War, Strife, Fury—and Death.

In a post-credit scene, Lilith is seen being berated by a being who is completely in shadow (refereed to by Lilith as 'my Prince'), angered that humanity has been restored and the Nephilim are lost forever. Lilith says she awaits punishment, but the entity states that "[she] will get no pleasure from it this time". The screen fades as Lilith screams in agony.