Background: Co-supplementation of unsaturated fatty acids (FAs) with saturated FAs may decrease the adverse effects of saturated FA-induced lipotoxicity. The objective of the present study was to evaluate the effect of palmitoleic acid (unsaturated fatty acid) on palmitic acid (saturated fatty acid) induced lipotoxicity criteria in the primary culture of adult rat cardiomyocytes. Methods: Cells were treated with 0.5 mM palmitic acid, palmitoleic acid, palmitic + palmitoleic acids or remained untreated. The values of cellular triacylglycerol (TAG), diacylglycerol (DAG), DNA fragmentation and cellular viability were evaluated over 24 h, 48 h and 72 h time points. Results: Co-administration of palmitic and palmitoleic acids increased TAG values over 48 h and 72 h time points compared to the palmitic acid (34.37% and 62.79%, respectively; P <0.001), while decreased DAG values (18.85% and 29.42%, respectively; P <0.01). Moreover, palmitoleic acid decreased DNA fragmentation and increased viability when administrated with palmitic acid (P <0.05). Conclusion: Palmitoleic acid may be beneficial for diminishing adverse effects of palmitic acid in the rat cardiomyocytes through alterations in the molecule signaling levels.