Delta-6 desaturase (D6D), encoded by the Fads2 gene, catalyzes the first step in the conversion of alpha-linolenic acid to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ablation of D6D in whole-body Fads2-/- knockout (KO) mice results in an inability to endogenously produce EPA and DHA. Evidence supports a beneficial role for EPA and DHA on insulin-stimulated glucose disposal in skeletal muscle in the context of a metabolic challenge; however, it is unknown how low EPA and DHA levels impact skeletal muscle fatty acid composition and insulin signaling in a healthy context. The objective of this study was to examine the impact of ablating the endogenous production of EPA and DHA on skeletal muscle fatty acid composition, whole-body glucose and insulin tolerance, and a key marker of skeletal muscle insulin signaling (pAkt). Male C57BL/6J wild-type (WT), Fads2+/- heterozygous and KO mice were fed a low-fat diet (16% kcal from fat) modified to contain either 7% w/w lard or 7% w/w flaxseed for 24 weeks. No differences in total phospholipid (PL), triacylglycerol, or reactive lipid content were observed between genotypes. As expected, KO mice on both diets had significantly less DHA content in skeletal muscle PL. Despite this, KOmice did not have significantly different glucose or insulin tolerance compared to WT mice on either diet. Basal pAktSer473 was not significantly different between the genotypes within each diet. Ultimately, this study shows for the first time that the reduction of DHA in skeletal muscle is not necessarily detrimental to glucose homeostasis in otherwise healthy animals.