Transforming Growth Factor β1 (TGF-β1) is an important fibrogenic growth factor. In skeletal muscle it has been associated with a failure to repair damage and has been identified as an inhibitor of myogenesis [1]. Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid with anti-inflammatory properties, previously reported to protect against the damaging effects of TNF-α during skeletal muscle differentiation [2]. EPA also exerts anti-fibrogenic effects, some of which at least are mediated through suppression of TGF-β1, for example, blocking endothelin-1 induced cardiomyocyte hypertrophy [3] and prevention of rat liver fibrosis [4].We hypothesised that EPA may be able to prevent some of the inhibitory actions of TGF-β1 on skeletal muscle differentiation. C2C12 myoblasts were differentiated by culture in growth medium containing 2% horse serum (DM). Cells were treated with EPA (50μM) and TGF-β1 (1ng/ml) either independently, as a co-treatment or with EPA as a pre-treatment in DM for up to 168 hours. Myogenesis was evaluated morphologically by a myogenic index, by myotube metrics and by myosin heavy chain (MyHC) expression at 24 hour intervals. TGF-β1 delayed the appearance of multinucleated myotubes by approximately 72 hours compared to control, which confirms impediment of differentiation. The length and diameter of myotubes were significantly decreased in cells treated with TGF-β1 at all time points (p<0.05) compared to control. EPA, as a co-treatment or pre-treatment, significantly increased the width and length of myotubes (p<0.05) at all time points, however the myogenic index was only significantly increased (p<0.05) in cells pre-treated with EPA at 48 and 96 hours. In summary, EPA treatment protects against TGF-β1-associated reductions in myotube size. Further research will be required to determine whether EPA can also protect against the TGF-β1-associated delayed-onset of differentiation. Additional investigations will also aid in the elucidation of the mechanisms involved in this process.