Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID- 19 patients. Although the prevalent Mpro mutants in the SARS-CoV-2 Variants of Concern (e.g., Omicron) are still susceptible to nirmatrelvir, a rare mutation, H172Y, was found to significantly reduce nirmatrelvir’s inhibitory activity. As the selective pres- sure of antiviral therapy may favor resistance mutations, there is an urgent need to understand the effect of H172Y mutation on Mpro’s structure, function, and drug re- sistance. Here we report the molecular dynamics (MD) simulations as well as the measurements of stability, enzyme kinetics of H172Y Mpro, and IC50 value of nir- matrelvir. Simulations showed that mutation disrupts the interactions between the S1 pocket and N terminus of the opposite protomer. Intriguingly, a native hydrogen bond (H-bond) between Phe140 and the N terminus is replaced by a transient H- bond between Phe140 and Tyr172. In the ligand-free simulations, strengthening of this nonnative H-bond is correlated with disruption of the conserved aromatic stacking between Phe140 and His163, leading to a partial collapse of the oxyanion loop. In the nirmatrelvir-bound simulations, the nonnative H-bond is correlated with the loss of an important H-bond between Glu166 and nirmatrelvir’s lactam nitrogen at P1 position. These results are consistent with the newly reported X-ray structures of H172Y Mpro and suggest a mechanism by which the H172Y substitution perturbs the S1 pocket, leading to the decreased structural stability and binding affinity, which in turns explains the drastic reduction in catalytic activity and antiviral susceptibility.