Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid that is abundant in fish oil, and has anti-inflammatory or anti-obesity effects. However, the effects of EPA supplementation on mitochondrial content and dynamics (fusion and fission) in skeletal muscle has not been elucidated. We investigated the effects of EPA intake for 4 weeks on denervation-induced mitochondrial adaptation in mice skeletal muscle. ICR mice (male, 8 weeks old) were daily administrated olive oil (control) or EPA at a dose of 300 mg/kg body weight by gavage for 4 weeks. After 2 weeks of oil intake, mice underwent unilateral sciatic nerve transection surgery. The hindlimb without surgery served as the sham-operated control. Body and skeletal muscle weights did not differ between the control and the EPA groups. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and oxidative phosphorylation (OXPHOS) proteins were significantly decreased by denervation surgery. Denervation also induced the reduction of mitochondrial fusion molecules and the increase of mitochondrial fission molecules. Expression levels of PGC-1α and mitochondrial respiration protein MTCO1 were higher in the EPA group than in the control (olive oil) group. In addition, the EPA group contained higher levels of mitochondrial fusion protein OPA1. Our results suggest that EPA intake prevents the reduction of mitochondrial content and fusion proteins in denervated skeletal muscle.