The objective of the present study was to investigate the regulatory mechanism of protein S-nitrosylation on early postmortem beef muscle apoptosis. Beef semimembranosus (SM) muscles at 45 min postmortem were treated with nitric oxide (NO) donor, control (NaCl solution), or nitric oxide synthase (NOS) inhibitor for 24 h at 4 °C. Bcl-2 expression and mitochondrial membrane potential were significantly increased by the NO donor treatment at 6 h postmortem, while the NOS inhibitor group exhibited a lower Bcl-2 level and mitochondrial membrane potential in comparison with the control ( < 0.05). The cytochrome c expression analysis highlighted that NO donor incubation repressed cytochrome c release from mitochondria to the cytoplasm. Further, S-nitrosylation levels of caspase-3 and caspase-9 were elevated after incubation with the NO donor ( < 0.05), leading to decreased caspase-3 and caspase-9 activities ( < 0.05). The aforementioned findings imply that protein S-nitrosylation mediates postmortem apoptosis of beef SM through the mitochondrial apoptotic pathway.