Prolonged moderate-intensity exercise is characterized by a progressive reduction in carbohydrate oxidation and concomitant increase in fat oxidation. Pyruvate dehydrogenase (PDH) controls the entry of pyruvate into oxidative pathways and is a rate-limiting enzyme for carbohydrate metabolism. PDH is controlled by the activities of a kinase (PDK, inhibitory) and phosphatase (stimulatory). To test the hypothesis that increased PDK activity was associated with decreased PDH activity and carbohydrate oxidation during an acute exercise bout, seven recreationally active men completed 4 h of cycle exercise at 55% peak oxygen consumption. Muscle samples were obtained before and at 10 min and 4 h of exercise for the measurement of PDH activity and the extraction of intact mitochondria for the measurements of PDK activity and PDK-2 and PDK-4 protein expression. Carbohydrate oxidation was reduced ( P < 0.05) with exercise duration. Muscle glycogen content was lower ( P ≤ 0.05) at 4 h compared with rest and there was no change in muscle pyruvate content from 10 to 240 min during exercise (10 min: 0.28 ± 0.05; 240 min: 0.35 ± 0.09 mmol/kg dry muscle). PDH activity increased ( P < 0.05) above resting values at 10 min (2.86 ± 0.26 mmol·min−1·kg wet muscle−1), but was lower than 10 min after 4 h (2.23 ± 0.24 mmol·min−1·kg wet muscle−1) of exercise. PDK-2 and PDK-4 protein expression was not different from rest at 10 min and 4 h of exercise. PDK activity at rest averaged 0.081 ± 0.016 min−1, was similar at 10 min, and increased ( P < 0.05) to 0.189 ± 0.013 min−1 at 4 h. Although reduced glycolytic flux may have played a role in decreasing carbohydrate oxidation, the results suggest that increased PDK activity contributed to the reduction in PDH activity and carbohydrate oxidation late in prolonged exercise. The increased PDK activity was independent of changes in intra-mitochondrial effectors, and PDK-2 and PDK-4 protein content, suggesting that it was caused by a change in the specific activity of the existing kinases.