A major characteristic of type 2 diabetes mellitus (T2DM) is insulin resistance in skeletal muscle. A growing body of evidence indicates that oxidative stress which results from increased production of reactive oxygen and/or reactive nitrogen species (ROS/RNS) leads to insulin resistance, tissue damage and other complications observed in T2DM. It has been suggested that muscular free fatty acids (FFA) accumulation might be responsible for the mitochondrial dysfunction and insulin resistance seen in T2DM, although the mechanisms by which increased levels of FFA lead to insulin resistance are not well understood. To help resolve this situation, we report that saturated fatty acid palmitate stimulated the expression of the inducible form nitric oxide (NO) synthase (iNOS) and the production of ROS, and NO in L6 myotubes. Additionally, palmitate caused a significant dose-dependant increase in mtDNA damage, and a subsequent decrease in L6 myotube viability and ATP levels at concentration as low as 0.5 mM. Furthermore, palmitate induced apoptosis which was detected by DNA fragmentation, caspase-3 cleavage and cytochrome c release. N-acetyl cystein (NAc), a precursor compound for glutathione formation, aminoguanidine (A), an iNOS inhibitor, and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) (FeTPPS), a peroxynitrite inhibitor, all prevented palmitate-induced mtDNA damage and diminished palmitate-induced cytotoxicity. We conclude that exposure of L6 myotubes to palmitate induced mtDNA damage and triggered mitochondrial dysfunction which caused apoptosis. Additionally, our findings indicate that palmitate-induced mtDNA damage and cytotoxicity in skeletal muscle cells was caused by overproduction of peroxynitrite.