This study aimed to examine the effect of eicosapentaenoic acid (EPA) on skeletal muscle hypertrophy induced by muscle overload and the associated intracellular signaling pathways. Male C57BL/6J mice were randomly assigned to oral treatment with either EPA or corn oil for 6 weeks. After 4 weeks of treatment, the gastrocnemius muscle of the right hindlimb was surgically removed to overload the plantaris and soleus muscles for 1 or 2 weeks. We examined the effect of EPA on the signaling pathway associated with protein synthesis using the soleus muscles. According to our analysis of the compensatory muscle growth, EPA administration enhanced hypertrophy of the soleus muscle but not hypertrophy of the plantaris muscle. Nevertheless, EPA administration did not enhance the expression or phosphorylation of Akt, mechanistic target of rapamycin (mTOR), or S6 kinase (S6K) in the soleus muscle. In conclusion, EPA enhances skeletal muscle hypertrophy, which can be independent of changes in the AKT-mTOR-S6K pathway.