Polyunsaturated fatty acids (PUFAs) are important molecules for human health. We investigated the effects of three major omega-3 PUFAs on C2C12 myoblast proliferation. Both docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids decreased cell growth, whereas linolenic (ALA) acid did not, compared with the control. Cell cycle analysis showed that G(1) phase duration was increased markedly and S-phase duration was decreased by DHA and EPA. In contrast, there was no change in the G(1) or S-phase duration when the cells were treated with linolenic acid. To determine how DHA and EPA affected the cell cycle, cyclins and MAPK proteins were investigated. Western blotting and real-time quantitative PCR showed that DHA and EPA decreased cyclin E and CDK2 levels at both the protein and mRNA level. Also, MAPK phosphorylation levels were decreased by treatment with DHA and EPA. Our results indicated that different kinds of n-3 PUFA differentially affected myoblast cell proliferation. DHA and EPA decreased skeletal muscle cell proliferation through a mechanism involving MAPK-ERK.