Seahorse is been used as a traditional Chinese medicine and invigorant for many years. Among, the seahorse is considered to have anti-fatigue effects. However, few researches have demonstrated such an effect. This study, the anti-fatigue effect of a novel peptide purified (SH200) from the seahorse (Hippocampus abdominalis) was investigated in C57BL/6 mice. The effect of SH200 on exercise performance was assessed using a Rotarod test and clinical biochemical parameters. In addition, the expression levels of muscular-associated proteins were examined by western blot and immunohistochemical analysis. The results revealed that SH200 prolonged the running time of mice, reduced the expression of creatine, CK, LDH, l-lactate, Citrate, and increased the production serum glucose and FFA, hepatic glycogen, ATP, and SDH. Addition, SH200 also improved mitochondrial function and protected muscle fibers via AMMPK/PGC-1α signal pathway. Thus, the SH200 is a potential candidate natural agent that could be used to reduce physical fatigue.