Chronic peroxisome proliferator-activated receptor (PPAR)-α activation improves glucose metabolism in rodent models of insulin resistance and diabetes; however, PPAR-α deficiency was also reported to protect against high-fat diet (HFD)-induced insulin resistance. The aim of this study was to clarify the role of PPAR-α in the development of insulin resistance using PPAR-α knockout (KO) mice and wild-type controls (WT). Both WT and PPAR-α KO mice on HFD gained significantly more weight relative to chow-fed groups and displayed an increase in insulin levels and a decrease in adiponectin levels. Hyperinsulinemic-euglycemic clamp performed in the nonfasting state demonstrated that HFD caused a marked reduction in whole body, muscle, and white and brown adipose tissue glucose uptake in both WT and PPAR-α KO mice relative to chow-fed groups. Suppression of endogenous glucose production during the clamp was markedly blunted in both WT and PPAR-α KO HFD-fed mice, indicating liver insulin resistance. The magnitude of HFD-induced changes in the clamp parameters of insulin sensitivity was comparable in PPAR-α KO and WT mice. In conclusion, these data show that PPAR-α deficiency does not alter insulin sensitivity in mice fed normal chow diet and does not protect against HFD-induced insulin resistance as measured by hyperinsulinemic-euglycemic clamp in nonfasted state.