TGF-β1 is an important pro-fibrogenic growth factor. In skeletal muscle it has been has been identified as an inhibitor of myogenesis [1]. It has previously been determined that TGF-β1-induced transdifferentiation of C2C12 myoblasts involves Sphingosine kinase-1 (SK1) and altered Sphingosine-1-phosphate (S1P) receptor expression [2]. Ω-3 PUFA can attenuate TGF-β1 activities against myogenesis and reportedly interact with other antimyogenic pathways, such as TNFα-induced NFκB activation, via PPARγ [3]. It was therefore hypothesized that Ω-3 PUFA may prevent TGF-β1 induced transdifferentiation of C2C12 myoblasts by modulation of the SK1/S1P axis and/or via PPARγ. C2C12 myoblasts were differentiated by culture in growth medium containing 2% horse serum (DM). Cells were treated with 50μM eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), oleic acid (OA) or Linoleic acid (LA) and TGF-β1 (1ng/ml) either independently or as a co-treatment for 96 hours. PPARγ was specifically antagonized with GW9662 for 2hrs prior to withdrawal and replacement with treatment medium. Myogenesis was evaluated morphologically by a myogenic index, by myotube metrics and by myosin heavy chain (MyHC) expression and by qPCR of the myogenic markers MyoD and Myogenin. Fibrotic biomarkers αSMA and transgelin were evaluated by immunocytochemistry and qPCR. S1Pr1, S1Pr3 SK1 and PPARγ were examined by qPCR at 6hrs and 18hrs post-induction of differentiation. TGF-β1-induced decreases in MyHC protein expression/ myotube morphometrics, increased αSMA/ transgelin gene and protein expression and altered MyoD/ Myogenin gene expression were attenuated by co-treatment with EPA or DHA but not OA or LA. Furthermore, TGF-β1-induced increases in SK1 and S1Pr3:S1Pr1 were prevented by co-treatment with EPA or DHA in conjunction with TGF-β1. Both PUFAs separately reversed the TGF-β1-mediated decrease in PPARγ gene expression and significantly increased its gene expression above control levels. However, specific blockade of PPARγ did not prevent EPA or DHA blocking the effects of TGF-β1 on myogenesis. In conclusion the Ω-3 PUFAs, EPA and DHA, attenuate TGF-β1 induced transdifferentiation of C2C12 myoblasts by a mechanism involving the sphingosine kinase pathway but independently of PPARγ. Additionally this effect is considered unique to fish oils as it is not achieved by the non Ω-3 PUFAS, OA and LA.