Background: The cardiovascular benefit of fish oil in humans and experimental animals has been reported. Endothelin (ET)-1 is a well-known cardiac hypertrophic factor. However, while many studies link a fish oil extract, eicosapentaenoic acid (EPA), to cardiac protection, its effect (EPA) on cardiac hypertrophy and underlying mechanism(s) are unclear. Objective: The present study investigated whether EPA prevents ET-1-induced cardiomyocyte hypertrophy and potential pathways likely to underlie such an effect. Methods: Cardiomyocytes were isolated from neonatal rat heart, cultured for 3 days and then treated for 24 hours with either: vehicle-only (control), ET-1 (0.1 nM)-only or EPA (10 µM) pretreated with ET-1 (0.1 nM). The cells were harvested and analyzed for changes in: cardiomyocyte surface area, protein synthesis, expression of a cytoskeleton (-actinin) protein and cell signaling. Results: ET-1 induced a 97% increase in cardiomyocyte surface area, 72% increase in protein synthesis rate, elevated expression of alpha-actinin and signaling molecules [Transforming growth factor-1 (TGF-1), c-Jun N-terminal kinase (JNK) and c-Jun). Interestingly, development of these ET-1-induced cellular changes were attenuated by EPA. Moreover, the hypertrophied cardiomyocytes showed a 1.5-fold and 1.7-fold increase in ANP and BNP mRNA expression, the classical molecular markers of cardiac hypertrophy, respectively, changes that were also suppressed by EPA. Conclusion: Here we show that ET-1 induces cardiomyocyte hypertrophy and expression of hypertrophic markers, possibly mediated by JNK and TGF1 signaling pathways. These ET-1-induced effects were blocked by EPA, a major fish oil ingredient, suggesting that fish oil may have beneficial protective effects on cardiac hypertrophy.