Abstract: Objective To study effect of electrical stimulations of different intensities on mitochondrial oxidative stress in C2C12 myotubes and explore the molecular mechanisms. Methods After 7 days of differentiation, C2C12 myotubes were subjected to electrical stimulations (15 V, 3Hz, 30 ms) for 60, 120, or 180 min, and the morphological changes of muscular tubes were observed under inverted microscope. The levels of MDA and SOD activity of the cells were detected, and flow cytometry was used to detect mitochondrial reactive oxygen species (ROS) and membrane potential. Western blotting was used to detect the expression of PGC1, AMPK-Ser485, AMPK-Thr172, and AMPK in the cells. Results No significant changes occurred in the morphology of C2C12 myotubes in response to electrical stimulations. Electrical stimulation for 60 min resulted in significantly increased levels of MDA, AMPK-Ser485 and AMPK-Thr172 in the cells (P<0.05); simulations of the cells for 120 and 180 min caused significantly increased MDA, ROS, mitochondrial ROS, AMPK-Ser485 and PGC1 along with marked reduction of mitochondrial membrane potential (P<0.05). Conclusion Electrical stimulation significantly activates oxidative stress, and a longer stimulation time causes stronger mitochondrial oxidation. AMPK-Thr172 regulates oxidative stress induced by stimulations for a moderate time length, while AMPK-Ser485 and PGC1 function to modulate oxidative stress following prolonged stimulations.