Deterioration of neuromuscular junction (NMJ) integrity and function is causal to muscle atrophy and frailty, ultimately hindering quality of life and increasing the risk of death. In particular, NMJ is vulnerable to ischemia reperfusion (IR) injury when blood flow is restricted followed by restoration. However, little is known about the underlying mechanism(s) and hence the lack of effective interventions. New evidence suggests that mitochondrial oxidative stress plays a causal role in IR injury, which can be precluded by enhancing mitochondrial protein S-nitrosation (SNO). To elucidate the role of IR and mitochondrial protein SNO in skeletal muscle, we utilized a clinically relevant model and showed that IR resulted in significant muscle and motor nerve injuries with evidence of elevated muscle creatine kinase in the serum, denervation at NMJ, myofiber degeneration and regeneration, as well as muscle atrophy. Interestingly, we observed that neuromuscular transmission improved prior to muscle recovery, suggesting the importance of the motor nerve in muscle functional recovery. Injection of a mitochondria-targeted S-nitrosation enhancing agent, MitoSNO, into ischemic muscle prior to reperfusion reduced mitochondrial oxidative stress in the motor nerve and NMJ, attenuated denervation at NMJ, and resulted in accelerated functional recovery of the muscle. These findings demonstrate that enhancing mitochondrial protein SNO protects against IR-induced denervation at NMJ in skeletal muscle and accelerates functional regeneration. This could be an efficacious intervention for protecting neuromuscular injury under the condition of IR and other related pathological conditions.