A single bout of exercise increases glucose uptake and fatty acid oxidation in skeletal muscle, with a corresponding activation of AMP-activated protein kinase (AMPK). While the exercise-induced increase in glucose uptake is partly due to activation of AMPK, it is unclear whether the increase of fatty acid oxidation is dependent on activation of AMPK. To examine this, transgenic mice were produced expressing a dominant-negative (DN) mutant of α1-AMPK (α1-AMPK-DN) in skeletal muscle and subjected to treadmill running. α1-AMPK-DN mice exhibited a 50% reduction in α1-AMPK activity and almost complete loss of α2-AMPK activity in skeletal muscle compared with wild-type littermates (WT). The fasting-induced decrease in respiratory quotient (RQ) ratio and reduced body weight were similar in both groups. In contrast with WT mice, α1-AMPK-DN mice could not perform high-intensity (30 m/min) treadmill exercise, although their response to low-intensity (10 m/min) treadmill exercise was not compromised. Changes in oxygen consumption and the RQ ratio during sedentary and low-intensity exercise were not different between α1-AMPK-DN and WT. Importantly, at low-intensity exercise, increased fatty acid oxidation in response to exercise in soleus (type I, slow twitch muscle) or extensor digitorum longus muscle (type II, fast twitch muscle) was not impaired in α1-AMPK-DN mice, indicating that α1-AMPK-DN mice utilize fatty acid in the same manner as WT mice during low-intensity exercise. These findings suggest that an increased α2-AMPK activity is not essential for increased skeletal muscle fatty acid oxidation during endurance exercise.