The therapeutic use of polyunsaturated fatty acids (PUFA) in preserving insulin sensitivity has gained interest in recent decades; however, the roles of linoleic acid (LA) and α-linolenic acid (ALA) remain poorly understood. We investigated the efficacy of diets enriched with either LA or ALA on attenuating the development of insulin resistance (IR) in obesity. Following a twelve-week intervention, LA and ALA both prevented the shift towards an IR phenotype and maintained muscle-specific insulin sensitivity otherwise lost in obese control animals. The beneficial effects of ALA were independent of changes in skeletal muscle mitochondrial content and oxidative capacity, as obese control and ALA treated rats showed similar increases in these parameters. However, ALA increased the propensity for mitochondrial H₂O₂ emission and catalase content within whole-muscle, and reduced markers of oxidative stress (4-HNE and carbonyl content). In contrast, LA prevented changes in markers of mitochondrial content, respiratory function, H₂O₂ emission and oxidative stress in obese animals, thereby resembling levels seen in lean animals. Together, our data suggests that LA and ALA are efficacious in preventing IR but have divergent impacts on skeletal muscle mitochondrial content and function. Moreover, we propose that LA has value in preserving insulin sensitivity in the development of obesity; thereby challenging the classical view that n-6 PUFAs are detrimental.