The objective of the present study was to analyze the activation and expression patterns of upstream and downstream factors of PGC-1α to determine whether antioxidant (AO) supplementation inhibits mitochondrial biogenesis in skeletal muscles as an adaptation to endurance training, as well as to analyze changes in endurance capacity based on such findings. For this objective, 24 male Sprague-Dawley (SD) rats were allocated into 4 groups (vehicle-sedentary, V-Sed; vehicle-exercise, V-EX; antioxidant-sedentary, AO-Sed; antioxidant-exercise, AO-EX) of 6 rats each. The rats were then treated with vitamin C (500 mgkg⁻¹ body weightd⁻¹) or a placebo for 8 wk, and a swimming program was implemented in some rats during the last 4 wk of this period. Immediately after the last training session, blood was collected from the tail of each rat, and TBARS was measured to test the effect of vitamin C as an AO. As a result, increased oxidative stress from exercise was inhibited by vitamin C supplementation. Analysis of whether reduced oxidative stress by vitamin C supplementation also inhibited mitochondrial biogenesis within skeletal muscles showed that phosphorylation of p38 MAPK and AMPK, along with levels of PGC-1α, NRF-1, mtTFA, and mitochondrial electron transport enzymes, increased after endurance training in spite of vitamin C supplementation. Moreover, running time, distance, and total work increased significantly in the exercise group as compared to those in the sedentary group, regardless of vitamin C supplementation. These results indicate that mitochondrial biogenesis and endurance capacity increase as a result of endurance training, regardless of AO supplementation.