The aim of this study was to investigate the effect of a high-fat diet (HFD) on energy substrate utilization during long-term endurance exercise in mice. Male ICR mice (n = 32; 6 weeks old) were divided into two groups: low-fat diet (LFD, n = 16) and HFD (n = 16) and acclimatized to LFD or HFD feeding over 12 weeks. After 12 weeks, the two dietary groups were each divided into two groups with or without exercise (EX): LF-CON, LF-EX, HF-CON, and HF-EX groups. The exercise groups were trained to run on a treadmill for 12 weeks. At the end of the experimental protocol, energy metabolism in the whole body was measured at rest for 24 h and during exercise for 1 h using respiratory gas analysis. Furthermore, molecules involved in skeletal muscle fat metabolism were analyzed. Substrate utilization for energy metabolism in the whole body indicated that fat utilization was high in HFD intake. Notably, when HFD intake and exercise were combined, fat utilization was markedly increased during endurance exercise. In contrast, exercise showed no effect when combined with LFD intake. The gene expressions of Fat/Cd36, Fatp1, Fabp-pm, and Cpt1 were upregulated by HFD intake, with Fat/Cd36 and Cpt1 considerably elevated during long-term endurance exercise. In contrast, exercise showed no effect when combined with LFD intake. These results suggest that HFD intake effectively increased fat utilization as an energy substrate during long-term endurance exercise.KEYWORDS: endurance exercise fat oxidation fatty acid transporter high-fat diet skeletal muscle *These authors contributed equally to this work.