This study was designed to evaluate the effect of dietary conjugated linoleic acid (CLA) on endurance exercise and availability of metabolic substrates in mice. Four-week-old male BALB/c mice were randomized to a control group (normal diet) and a CLA group (normal diet + 1% CLA). Each animal group received its respective diet for 10 weeks and was subjected to forced running on a treadmill system to estimate their maximum endurance capacity at the end of the experiment. All mice were immediately sacrificed after prolonged exercise, and the major metabolic substrates were measured in serum and liver. The body weights of mice in the CLA group were lower than those of the control group after the 10 weeks. The maximum running time in CLA-fed mice was significantly longer, by 26%, compared to that of the control mice. Dietary CLA decreased the serum concentrations of triglycerides, nonesterified fatty acids, and urea nitrogen and significantly reduced the consumption of liver glycogen. The present study demonstrated that dietary CLA enhances the endurance capacity of mice by increasing fat utilization and reducing the consumption of stored liver glycogen as substrates for energy metabolism.