Excess intramuscular lipids are thought to play a causal role in the pathogenesis of insulin resistance; thus, strategies aimed at reducing this lipid burden offer therapeutic potential. Peroxisomes provide an intriguing option to meet this goal as they have the ability to process a broad spectrum of lipid species. As such, the purpose of this study was to determine if skeletal muscle peroxisomes protect against lipid-induced insulin resistance. To test this hypothesis, we developed a muscle-specific peroxisome-deficient mouse model by deleting Pex5 in skeletal muscle (Pex5m-/-) and weaned them onto a moderate fat (25%) diet to which they had ad libitum access for 20 weeks. Pex5m-/- mice had similar body weight and composition when compared to Pex5fl/fl littermate controls, but exhibited impaired glucose tolerance. This impairment in whole body glucose homeostasis was associated with a 25-30% decrease in mitochondrial function in Pex5m-/- mice, suggesting that peroxisomes helped protect against mitochondrial dysfunction in skeletal muscle. To further test this potential link between peroxisomes and mitochondria in skeletal muscle we analyzed peroxisomal adaptations in a murine model where mitochondrial fatty acid oxidation was limited via muscle-specific deletion of the rate-limiting enzyme of mitochondrial lipid entry, Cpt1b (Cpt1bm-/-). As expected, the limitation in mitochondrial fatty acid entry in Cpt1bm-/- mice resulted in a compensatory upregulation of peroxisomes which helped maintain adequate lipid oxidation. These adaptations were associated with robust improvements in glucose tolerance in Cpt1bm-/- mice. To test the importance of peroxisomal adaptations in mediating these improvements, we developed a Pex5:Cpt1bm-/- mouse model to prevent peroxisomal compensation in the Cpt1bm-/- strain. Results from these studies revealed that skeletal muscle fatty acid oxidation was severely compromised in Pex5:Cpt1bm-/- and that abrogation of peroxisomal function prevented the improvement in glucose tolerance that was incurred by muscle-specific Cpt1b deletion alone. Overall these results suggest that peroxisomes in skeletal muscle play an important role in maintaining whole body glucose homeostasis and that this is likely at least partially attributed to the fact that peroxisomes provide an alternate route for lipid disposal which limits mitochondrial lipid overload.