Accumulation of excess saturated free fatty acids such as palmitate (PAL) in skeletal muscle leads to reductions in mitochondrial integrity, cell viability and differentiation. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) counteract PAL-induced lipid accumulation. EPA and DHA, as well as the n-3 PUFA docosapentaenoic acid (DPA), may therefore mitigate PAL-induced lipotoxicity to promote skeletal muscle cell survival and differentiation. C2C12 myoblasts were treated with 50 μM EPA, DPA, or DHA in the absence or presence of 500 μM PAL for 16 h either prior to myoblast analysis or induction of differentiation. Myoblast viability and markers of apoptosis, endoplasmic reticulum (ER) stress and myotube differentiation capacity were investigated using fluorescence microscopy and immunoblotting. High-resolution respirometry was used to assess mitochondrial function and membrane integrity. PAL induced cell death via apoptosis and increased protein content of ER stress markers BiP and CHOP. EPA, DPA, and DHA co-treatment maintained cell viability, prevented PAL-induced apoptosis and attenuated PAL-induced increases in BiP, whereas only DPA prevented increases in CHOP. PAL subsequently reduced protein content of the differentiation marker myogenin and inhibited myotube formation, and all n-3 PUFAs promoted myotube formation in the presence of PAL. Furthermore, DPA prevented PAL-induced release of cytochrome c and maintained mitochondrial integrity. These findings demonstrate the n-3 PUFAs EPA, DPA and DHA elicit similar protective effects against PAL-induced impairments in muscle cell viability and differentiation. Mechanistically, the protective effects of DPA against PAL lipotoxicity are attributable in part to its ability to maintain mitochondrial respiratory capacity via mitigating PAL-induced loss of mitochondrial membrane integrity.