Abstract The reaction mechanism of the covalent binding of complement component 3 (C3) with antigen, which significantly contributes to the immune system by enhancing the phagocytosis of phagocytes, is examined by density functional theory (B3LYP). The covalent binding reaction takes place by the cleavage of the hydroxy O–H bond of the antigen on the thioester bond of the C3b formed by the activation of C3. We performed the calculations assuming a mechanism II that mimics that of serine protease in addition to the conventional mechanism I. Our calculations suggest that the conventional mechanism I proceeds with little participation of a His residue contrary to the previous consensus. Mechanism I, which has a large energy barrier of more than 30 kcal mol−1, is not realistic. On the other hand, in mechanism II, the energy barrier is about 10 kcal mol−1, due to an efficient mediation of a His residue by the collaboration with a neighboring Glu residue. Therefore, the covalent binding reaction of the activated C3b with the antigen was thought to proceed by mechanism II we proposed rather than by the conventional mechanism I.