Kirei Kotomine is a central figure in the Fifth Holy Grail War as the war's appointed supervisor from the Church, though his role is deeply compromised by his own secret participation and agenda. Standing at 193 centimeters tall with a muscular build, he has short brown hair and brown eyes in his younger years, though by the time of the Fifth Holy Grail War, his hair has grown longer and he wears a dark purple coat over his priestly vestments. He is the son of Risei Kotomine, a high-ranking priest within the Holy Church's Eighth Sacrament. Despite his position as a man of faith, Kirei is fundamentally broken in a way he cannot repair.

At his core, Kirei is a man born with a fundamental defect. He is incapable of finding joy in the things that normal people consider beautiful or good. Instead, he derives a sense of satisfaction and happiness from the suffering and despair of others. This is not a choice or a gradual corruption, but an innate part of his being that he struggled against for much of his life. During his youth, he desperately tried to find a cure for this emptiness. He sought answers through his faith, rigorous training, marriage, and raising a family. He married a woman named Claudia Hortensia, who was ill and had only a few years to live, hoping that domestic life would fill the void. However, even as she sacrificed herself to prove he was capable of love, Kirei found himself disappointed not by her death, but because he had not been the one to kill her. This realization led him to abandon his attempts at normalcy.

His motivations shift dramatically during the Fourth Holy Grail War. Initially serving as a Master and disciple to Tokiomi Tohsaka under his father's orders, Kirei is a hollow man going through the motions. It is through his interactions with his Servant, the heroic spirit Gilgamesh, that he finally accepts his true nature. Gilgamesh, amused by Kirei's inner turmoil, encourages him to stop fighting his desires. The climax of the Fourth War, which results in a massive fire that devastates a section of Fuyuki City, is a moment of epiphany for Kirei. Witnessing the widespread death and anguish brings him genuine joy for the first time, cementing his path. His ultimate goal in the Fifth Holy Grail War is not to make a wish for himself, but to witness the birth of Angra Mainyu, the corrupted essence within the Grail. As a being he believes is born of pure evil like himself, he hopes that by seeing its nature, he can finally understand his own existence.

In the story, Kirei plays a dual role. Publicly, he is the priest of the Kotomine Church in Fuyuki City, tasked with overseeing the Holy Grail War, offering sanctuary to Masters who have lost their Servants, and ensuring the secret of magic is preserved. Privately, he is the war's most flagrant cheater. He is the secret Master of both Lancer and Gilgamesh. He steals Lancer from Bazett Fraga McRemitz, the original summoner, by ambushing her and taking her Command Spells. He also maintains his contract with Gilgamesh from the previous war, sustaining the Servant with the life force of orphaned children kept in the church's basement.

His key relationships define his actions. His relationship with Gilgamesh is one of mutual understanding and partnership rather than a typical Master-Servant bond; Gilgamesh is the one who helped Kirei embrace his desires. He is the guardian of Rin Tohsaka, having taken her in after killing her father, Tokiomi. He trains her in martial arts and magecraft, all while hiding his betrayal. He serves as a dark mirror to the protagonist, Shirou Emiya. Both men are described as being fundamentally broken, but while Shirou survived a great fire and dedicated his life to saving others as a borrowed ideal, Kirei survived the same fire and dedicated himself to causing suffering as his true nature.

Kirei undergoes very little development throughout the Fifth War because he has already found his answer. His change happened a decade prior. In the story, he is a static and formidable obstacle. In the Heaven's Feel route, his character reaches its final conclusion. Having lost the use of his heart, which was replaced by the Grail's corrupting mud, he exists in a state of living death. In the final battle, he fights Shirou not with magic or Servants, but in a brutal hand-to-hand brawl, representing a pure clash of their opposing ideals. He finally dies, having found contentment in his final confrontation.

Kirei possesses a diverse and dangerous set of abilities. Despite not being born into a magus family, he was born with Magic Circuits, allowing him to use magecraft. He is a master of Bajiquan, a Chinese martial art that focuses on explosive, close-range strikes that can damage internal organs, capable of shattering a thick tree trunk with a single blow. As a former Executor of the Church, he wields Black Keys, long, thin blades used as projectile weapons that are highly effective against spiritual beings. He is an expert in spiritual healing, a rare form of magecraft that mends the soul rather than the body, and he uses this skill to perform surgeries like transplanting a Servant's arm or removing magical parasites. His signature spell is the Baptism Sacrament, a Church ritual that acts as a powerful exorcism, capable of instantly destroying wraiths and severely damaging even ancient spirits like Zouken Matou. He carries an immense number of Command Spells on his right arm, inherited from his father, which he can use to empower his own physical abilities or as a one-time source of magical energy. His physical condition is at its peak during the Fourth Holy Grail War, and by the Fifth War, his combat ability has declined from his prime.