Abstract: Objective:To investigate the effects of apelin supplement during aerobic training on AMPK activation and mitochondrial energy metabolism in skeletal muscle. Methods: In the cell experiments, the AMPKα specific small interfering RNA (AMPKαsiRNA) or a negative control interfering RNA (Control siRNA) were transfected into mice skeletal muscle C2C12 cells by Lipofectamine. Starvation treatment was performed for 6 hours after 48 hours transfection, and then cells were incubated with apelin-13 or PBS for 6 hours in complete medium to observe the changes in mitochondrial basal respiration, ATP production, and respiratory function. In the animal experiments, forty 8-week-old C57BL/6J mice were randomly divided into four groups (n=10 in each group) : sedentary without apelin treatment, sedentary with apelin treatment, exercise training without apelin treatment and exercise training with apelin treatment group. The apelin treatment groups were injected intraperitoneally with apelin-13 at 0.1 μmol/kg/day for 4 weeks. The exercise groups were trained 6 days/week, 1 hour/day for 4 weeks by running on a treadmill at 75% VO2 max. 48 hours after the last session of exercise training, the quadriceps were collected. The protein expressions of apelin, APJ, AMPKα, p-AMPKα (Thr172) and COX IV in skeletal muscles was measured by Western Blotting. Results: 1) In the cell experiments, the basal respiration rate, mitochondrial ATP production and maximum respiration rate were significantly increased in the Control siRNA+apelin group compared with the Control si RNA+PBS group, but the mitochondrial ATP production and maximum respiration rate were significantly decreased in the AMPK siRNA+apelin group compared with the Control siRNA+apelin group. 2) In the animal experiments, the protein expression of apelin, APJ, COX IV and the p-AMPKα/AMPKα ratio in skeletal muscles were significantly increased in the apelin treatment groups compared with no apelin treatment groups. Conclusion: The exogenous supplementation of apelin was significantly increased the AMPK-mediated mitochondrial respiratory function in C2C12 cells, and the apelin supplement during aerobic training was also significantly increased the apelin/APJ, COX IV protein expression and the p-AMPKα/AMPK ratio in skeletal muscles. The results suggested that the combination of aerobic exercise training with apelin supplement might activates the apelin-AMPK pathway and improves the mitochondrial energy metabolism in skeletal muscles.