The aim of the current research was to examine the influence of nitric oxide (NO) on calpain activation, protein proteolysis, and oxidation in post-mortem pork. Five longissimus muscles were removed from carcass after slaughter, and samples were incubated with water, nitric oxide synthase (NOS) inhibitor, or NO donor for 24 h at 4 °C. The samples were taken out and then stored under 4 °C for 1, 4, and 7 d. Results showed that autolysis of μ-calpain increased by incubation with NOS inhibitor after storage for 1 d (P<0.05). Degradation of titin and nebulin increased by treatment of NOS inhibitor among three treatments (P<0.05). Higher levels of protein oxidation were observed after samples incubated with NO donor than treatment of NOS inhibitor (P<0.05). These data indicated that NO could participate in regulating calpain activation and its proteolysis activity during post-mortem aging.