In vitro studies of isolated skeletal muscle have shown that oxidative stress is limiting with respect to contractile function. Mitochondria are a potential source of muscle function-limiting oxidants. To test the hypothesis that skeletal muscle-specific mitochondrial oxidative stress is sufficient to limit muscle function, we bred mice expressing Cre recombinase driven by the promoter for the inhibitory subunit of troponin (TnIFast-iCre) with mice containing a floxed Sod2 (Sod2^fl/fl) allele. Mn-SOD activity was reduced by 82% in glycolytic (mainly type II) muscle fiber homogenates from young TnIFastCreSod2^fl/fl mice. Furthermore, Mn-SOD content was reduced by 70% only in type IIB muscle fibers. Aconitase activity was decreased by 56%, which suggests an increase in mitochondrial matrix superoxide. Mitochondrial superoxide release was elevated more than twofold by mitochondria isolated from glycolytic skeletal muscle in TnIFastCreSod2^fl/fl mice. In contrast, the rate of mitochondrial H₂O₂ production was reduced by 33%, and only during respiration with complex II substrate. F₂-isoprostanes were increased by 36% in tibialis anterior muscles isolated from TnIFastCreSod2^fl/fl mice. Elevated glycolytic muscle-specific mitochondrial oxidative stress and damage in TnIFastCreSod2^fl/fl mice were associated with a decreased ability of the extensor digitorum longus and gastrocnemius muscles to produce contractile force as a function of time, whereas force production by the soleus muscle was unaffected. TnIFastCreSod2^fl/fl mice ran 55% less distance on a treadmill than wild-type mice. Collectively, these data suggest that elevated mitochondrial oxidative stress and damage in glycolytic muscle fibers are sufficient to reduce contractile muscle function and aerobic exercise capacity.