Kakuzu first appeared with his partner, Hidan, in their pursuit of the Two-Tailed Monster Cat's jinchūriki, Yugito Nii, who revealed she allowed herself to be cornered, in order to spring a trap on them and eliminate them herself. Yugito then transformed into her full demon form, a large flaming cat, but the Akatsuki duo still managed to defeat her, and gave her body to Zetsu, before they continued to the Land of Fire.
On their way, Kakuzu decided to attack the Fire Temple, in order to collect the bounty on the head monk, Chiriku, who was listed in the bingo book with a thirty million ryō bounty. They slaughtered most of the other monks during the event, but one escaped to warn Konoha of the incoming threat.
Upon receiving the notice, Tsunade assigned the Nijū Shōtai to track down and eliminate Kakuzu and Hidan. Meanwhile, Kakuzu took Chiriku's body to a bounty station hidden in a bathroom, while Hidan chose to wait outside. When Kakuzu finally left the bathroom after counting the money, he found a Nijū Shōtai team, led by Asuma Sarutobi, attacking Hidan. He attacked Shikamaru Nara in order to free Hidan, but was forbidden from further participation in the battle by the overconfident Hidan himself. Kakuzu complied, but insisted that he would get to have the thirty-five million ryō head of Asuma afterwards. In the ensuing battle, Hidan managed to ingest Asuma's blood to start his curse jutsu ceremony on him, but Shikamaru was able to stop him with his Shadow Possession Technique. When Asuma managed to decapitate Hidan, Kakuzu finally intervened, and reattached Hidan's head back to his neck using the threads in his body. Kakuzu then fought with Kotetsu and Izumo, easily holding the two off, while Hidan dealt a fatal blow to Asuma. Their battle was interrupted by the appearance of another Nijū Shōtai team, but they easily fought them off, until they were summoned by the Akatsuki leader to seal the Two-Tails. Hidan and Kakuzu retreated to a secluded area, where they spent three days sealing the Two-Tails (and in the manga, another three days sealing the Three-Tailed Giant Turtle).
After the sealing was complete, Kakuzu and Hidan set out on their search for the Nine-Tailed Demon Fox's jinchūriki (Naruto Uzumaki), only to be cut off by Team 10 (with Kakashi Hatake as the leader in place of Asuma). Shikamaru used Asuma's chakra knives to capture both the Akatsuki members in place with a trick, but Kakuzu had discovered the trick beforehand, and, using a separated arm, broke free. Because of his ability to harden his skin, Kakuzu proved to be largely invulnerable to subsequent attacks. Kakashi, however, was able to analyse Kakuzu's hand seals, and pierced Kakuzu's heart with his Lightning Cutter, exploiting the weakness Kakuzu's earth-based jutsu had against lightning-based ones.
While this would normally be fatal, Kakuzu had five hearts (now four) in his possession, and thus recovered fairly quickly, sending a counterattack to Kakashi. Removing his cloak, he revealed four masks on his back, one of which was now broken by Kakashi. Each mask contained a heart, the broken one having given its heart up to keep Kakuzu alive. The four masks broke free from his back into separate entities, with the broken one dissolving after doing so. Each mask was able to attack with a specific and powerful elemental attack, making Kakuzu almost too much for Team 10 to handle. While Kakuzu set his sights on replacing his lost heart with Kakashi's, Shikamaru dragged Hidan away from the battlefield into some woods, with his Shadow Possession Technique, for a one-on-one fight.
Ignoring the other two for the most part, Kakuzu focused on taking Kakashi's heart. Though Kakashi was unable to handle Kakuzu and his mask creatures, he was saved when Shikamaru tricked Hidan into using some of Kakuzu's blood in his curse ritual, which Kakashi had collected in a capsule earlier when he pierced Kakuzu's chest, and had handed it over to Shikamaru. This resulted in Hidan unintentionally destroying Kakuzu's main body's heart. Seeing this, the Lightning mask allowed its heart to be absorbed by Kakuzu's body, bringing him back to life. In addition to that, Kakuzu also had the two other Fire and Wind masks to combine with him, making him even more powerful. He then caught the rest of Team 10 with his threads, hoping to replenish his stock.
Naruto striking Kakuzu with his Wind Release: Rasenshuriken.
Team 10 was saved by the arrival of Team 7, among them being Naruto, who decided to fight Kakuzu himself, who at first did not view Naruto as a threat. This changed when Naruto unveiled his newly developed technique, Wind Release: Rasenshuriken, which Kakuzu recognized to be dangerous if it ever connected with him. Naruto's inability to maintain the technique for more than a few seconds put Kakuzu at ease, especially since its failure almost allowed him to kill Naruto (although Yamato and Kakashi saved him), but decided to fight in his long-range mode just to be safe. As Naruto tried the Rasenshuriken once again with his shadow clones, Kakuzu attempted to crush Naruto with his threads from a distance, but was surprised to discover that it was a shadow clone that he had destroyed, and, subsequently, the real Naruto shot up behind him, using the technique effectively this time.
Left with no usable hearts after being battered by Naruto's technique, Kakuzu lamented being defeated by those he considered "kids". Kakashi assured Kakuzu that the previous generation being surpassed by the next was perfectly natural, and one of the "never-ending cycles of life". Kakashi then finished him off with a Lightning Cutter.
Kakuzu's corpse was later taken back to Konoha to be autopsied by Tsunade in regards of the Rasenshuriken's capabilities, and it revealed that Kakuzu was damaged at a cellular level by it.
