Itachi awakened the Mangekyō Sharingan after the suicide of Shisui Uchiha. Its design was three spiralling curves around the pupil. With his left eye, he could use Tsukuyomi, a genjutsu that trapped opponents' minds in an illusory world, altering their perception of time to make seconds of torture seem like days. With his right eye he could use Amaterasu, igniting whatever he looked at with black flames that would burn anything, including fire itself. He had the ability to extinguish Amaterasu, but never displayed the sort of shape transformation that Sasuke later would.

Having awakened the Mangekyō in both his eyes, Itachi could also use Susanoo. With its simplest manifestations, he could produce extra arms or bones to improve his options in a fight. When used in full, Itachi was surrounded by a spectral warrior that would protect him from all damage, even jutsu as powerful as Kirin. In addition to the chakra swords and Yasaka Magatama common to all Susanoo, Itachi's Susanoo wielded the Sword of Totsuka – an ethereal sword with the ability to seal any person it pierced into its gourd—hilt – and the Yata Mirror – a shield that was said to reflect any attack by changing its chakra nature to counterbalance an attack. The simultaneous use of both weapons made Itachi's Susanoo essentially invincible.

Using any of these techniques required vast amounts of chakra to perform. If he used these techniques too often in a short span of time, Itachi would become so fatigued that he would deactivate his Sharingan altogether. Every time he used his Mangekyō Sharingan his eyesight deteriorated, a process that would progressively worsen on each occasion. Early uses of the Mangekyō would only cause his eyes to bleed, but by the end of his life he reached the point where he was nearly blind, most things looking to him like mere blurs. He does not suffer these shortcomings after his reincarnation and can use all his Mangekyō techniques without problems.