Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction-mediated glucose transport. Repeated contractions increased 2-deoxyglucose (2-DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast-twitch) muscle. N-Acetylcysteine (NAC), a non-specific antioxidant, inhibited contraction-mediated 2-DG uptake by ∼50% (P < 0.05 versus control values), but did not significantly affect basal 2-DG uptake or the uptake induced by insulin, hypoxia or 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, which mimics AMP-mediated activation of AMP-activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction-mediated 2-DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+-dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a ∼25% higher rate of contraction-mediated 2-DG uptake versus muscles from wild-type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction-mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation ∼10-fold, and NAC blocked ∼50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction-mediated activation of glucose transport in fast-twitch muscle.