High doses of the antioxidant vitamin C prevent the increases in skeletal muscle mitochondrial biogenesis after exercise training. Since exercise training effects rely on the acute stimulus of each exercise bout, we examined whether vitamin C supplementation also attenuates the increases in skeletal muscle metabolic signaling and mitochondrial biogenesis in response to an acute exercise bout. Male Sprague-Dawley rats performed 60 min of treadmill running (27 m/min, 5% grade) or remained sedentary. For 7 days before this, one-half of the rats received water containing 500 mg/kg body wt vitamin C. Acute exercise significantly (P < 0.05) increased the phosphorylation of p38 MAPK, AMP-activated kinase-α, and activating transcription factor (ATF)-2 and the ratio of oxidized to total glutathione (GSSG/TGSH) in the gastrocnemius. However, vitamin C had no effect on these increases. Similarly, vitamin C did not prevent the exercise-induced increases in peroxisome proliferator-activated receptor-γ coactivator-1α, nuclear respiratory factor (NRF)-1, NRF-2, mitochondrial transcription factor A, glutathione peroxidase-1, MnSOD, extracellular SOD, or glucose transporter 4 (P < 0.05) mRNA after exercise. Surprisingly, vitamin C supplementation significantly increased the basal levels of GSSG/TGSH, NRF-1, and NRF-2 mRNA and basal ATF-2 phosphorylation. In summary, despite other studies in rats showing that vitamin C supplementation prevents increases in skeletal muscle mitochondrial biogenesis and antioxidant enzymes with exercise training, vitamin C had no affect on the acute exercise-induced increases of these markers.