Protein Arginine Deiminase 4 (PAD4) catalyzes the citrullination of the peptidylarginine via two successive stages: deimination and hydrolysis. Herein, by employing state-of-the-art BornOppenheimer ab initio QM/MM molecular dynamics simulations with umbrella sampling method, we have characterized the catalytic mechanism of the hydrolysis reaction: first the nucleophilic attack of a water molecule to the C ζ of the thiouronium intermediate yields a stable tetrahedral intermediate, and then the S-C ζ bond breaks to generate the final product, citrulline. Throughout the hydrolysis reaction, His471 and Asp473 play pivotal catalytic roles by first enhancing the nucleophilic ability of the active water through forming shorter and low-barrier hydrogen bonds, and then by serving as proton-accepting groups to deprotonate the water molecule, which is consistent with experimental findings. At the transition state, the spontaneous proton transfer among the reactive water, His471 and Asp473 has been observed. The determined overall free energy barrier for this hydrolysis stage is 16.5 kcal·mol -1 , which is lower than the barrier of 20.9 kcal·mol -1 for the deimination stage determined previously with the same computational approach [J. Phys. Chem. B, 113, 12750-12758 (2009)]. Thus the rate-determining step of the PAD4 catalyzed citrullination is the first step of the deimination. Our current work further demonstrates the strength and applicability of the ab initio QM/MM MD approach in simulating enzyme reactions.