Itachi debuts in Part I after the death of the , Konoha's protector, by infiltrating the village with Kisame. After drawing the attention of Konoha's forces Itachi reveals that, as part of Akatsuki's goal to capture the nine powerful , they are after Naruto Uzumaki for the sealed within him. During a scuffle with Konoha's ninja they discover that Naruto has left the village, and upon tracking him down find that he is under the protection of . Itachi sets up a distraction to draw Jiraiya away from Naruto and the two try to capture him. Sasuke, having heard that Itachi was back, intervenes, and Jiraiya soon returns as well. After briefly tormenting Sasuke for still being weak, Itachi and Kisame leave to avoid capture by Jiraiya. After Itachi's death in Part II, this entire visit to Konoha is revealed to have only been to make sure that the Third's death did not cause Konoha to forget their vow to protect Sasuke.
Itachi makes a brief appearance at the start of Part II, distracting Naruto and the rest of while Akatsuki seals one of the tailed beasts it has captured. Later, Itachi learns that Sasuke has absorbed , a man who defected from Konoha for in order to get strong enough to kill Itachi. Knowing that a stronger Sasuke is now coming for him, Itachi meets with Naruto. After his death he is revealed to have given some of his power to Naruto to be used to protect Sasuke and Konoha in case Sasuke ever attacks the village. Sasuke ultimately tracks him down and they have their final battle, during the course of which he uses his Mangekyo Sharingan to push Sasuke to his limits. In doing so, Orochimaru is drawn out from Sasuke's body, and Itachi seals him away to keep him from Sasuke before he dies of disease.
As his final act, Itachi implants his Amaterasu within Sasuke, meant as a failsafe that would kill Madara in case he ever approached Sasuke. Since this surprises Sasuke, Madara explains the truth of Itachi's past, what is claimed to be an unfounded mistrust of Madara, and that, even though Itachi had been dying of disease, he kept himself alive with drugs so that he could die by Sasuke's hands. Now knowing his long-hated brother to have led a tragic life because of Konoha's orders, Sasuke resolves to destroy the village, even though Itachi had gone to lengths to protect it.