The catalytic mechanism of carboxypeptidase A (CPA) for the hydrolysis of ester substrates is investigated using hybrid quantum mechanical/molecular mechanical (QM/MM) methods and high-level density functional theory. The prevailing mechanism was found to utilize an active-site water molecule assisted by Glu270, and this so-called promoted-water pathway is similar to that in the CPA catalyzed proteolytic reaction (D. Xu and H. Guo, J. Am. Chem. Soc. 2009, 131, 9780). On the other hand, our simulations indicated the existence of an alternative pathway due to direct nucleophilic attack of Glu270 on the scissile carbonyl carbon. This so-called nucleophilic pathway, which is not viable in proteolytic reactions, leads to a stable acyl-enzyme complex. However, the nucleophilic pathway is nonproductive as it is blocked by a high barrier in the deacylation step. On the basis of results reported here and in our earlier publication, a unified model is proposed to account for nearly all experimental observations concerning the catalysis of CPA.