The peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)-activated signal transduction pathway has previously been shown to stimulate mitochondrial biogenesis in skeletal muscle in response to endurance exercise. In vitro data indicate that PGC-1α signaling may be sensitive to reactive oxygen species (ROS) but its role in vivo is not clear. The objectives of this study were (1) to investigate whether the PGC-1α pathway could be activated by a single bout of anaerobic exercise in rats, wherein a major portion of ROS was generated via the cytosolic xanthine oxidase (XO), and (2) to examine whether allopurinol (ALP), a specific XO inhibitor, would attenuate PGC-1α expression and signaling owing to decreased ROS generation. Female Sprague–Dawley rats were randomly divided into three groups: (1) subjected to sprinting on a treadmill at 35 m/min, 15% grade, for 3 min followed by 3 min slow running at 15 m/min, 0% grade, repeated until exhaustion (88 ± 4 min; Exer; N = 9); (2) subjected to the same exercise protocol (88 ± 4 min) but injected with two doses of ALP (0.4 mmol/kg, ip) 24 and 1 h before the experiment (Exer+ ALP; N = 9); and (3) rested control (C; N = 9). Exercise increased XO activity and ROS generation in the Exer rat vastus lateralis muscle ( P < 0.05), whereas the Exer+ ALP group displayed only 7% XO activity and similar ROS level compared with the C group. PGC-1α protein content showed a 5.6-fold increase ( P < 0.01) in Exer vs C, along with a 200% ( P < 0.01) increase in both nuclear respiratory factor (NRF)-1 and mitochondrial transcription factor A (Tfam) content. ALP treatment decreased PGC-1α, NRF-1, and Tfam levels by 45, 19, and 20% ( P < 0.05), respectively. Exercise doubled the content of the phosphorylated cAMP-responsive element-binding protein in the Exer group ( P < 0.01) and tripled phosphorylated p38 mitogen-activated protein kinase ( P < 0.01), whereas these effects were reduced by 60 and 30% ( P < 0.01, P < 0.05), respectively, in Exer+ ALP rats. Nuclear factor-κB binding and phospho-IκB content were also increased in Exer rats ( P < 0.01) and these increases were abolished by ALP treatment. The data indicate that contraction-activated PGC-1α signaling pathways in skeletal muscle are redox sensitive and that nonmitochondrial ROS play an important role in stimulating mitochondrial biogenesis.