The aim of this study was to determine if the decreased muscle and blood lactate during exercise with hyperoxia (60% inspired O₂) vs. room air is due to decreased muscle glycogenolysis, leading to decreased pyruvate and lactate production and efflux. We accomplished this by examining the five fates of muscle pyruvate: 1) pyruvate accumulation 2) oxidation via pyruvate dehydrogenase (PDH), 3) reduction to muscle lactate 4) lactate efflux, and 5) pyruvate efflux to estimate total pyruvate and lactate production during exercise. We hypothesized that 60% O₂ would decrease muscle glycogenolysis resulting in decreased pyruvate and lactate contents leading to decreased muscle pyruvate and lactate release with no change in PDH activity (pyruvate oxidation). Seven active male subjects cycled for 40 min at 70% VO_2peak on two occasions while breathing 21 or 60% O₂. Arterial and femoral venous blood samples and blood flow measurements were obtained throughout exercise, and muscle biopsies were taken at rest and following 10, 20 and 40 min of exercise. Muscle glycogenolysis was reduced by 16% with hyperoxia (267 ± 19 vs. 317 ± 21 mmol glycosyl units^.kg^-1 dw), which translated into a significant 15% reduction in total pyruvate production over the 40 min exercise period. The decreased pyruvate production with hyperoxia had no effect on PDH activity (pyruvate oxidation) (60%: 3.02 ± 0.50 vs. 21%: 3.03 ± 0.39 mmol^.kg^-1 ww^.min^-1). Hyperoxia significantly decreased lactate accumulation (60%: 22.6 ± 6.4 vs. 21%: 31.3 ± 8.7 mmol^.kg^-1 dw), lactate efflux and total lactate production over 40 min of cycling. Hyperoxia also significantly decreased PCr degradation, attenuated the accumulations of Cr and calculated ADPf and AMPf and reduced blood epinephrine concentration by ~44% throughout 40 min of cycling, but had no effect on leg O₂ delivery, O₂ uptake, CO₂ production or RQ during exercise. The tighter metabolic match between pyruvate production, via decreased glycogenolysis, and oxidation, left less pyruvate for conversion to lactate resulting in a decrease in total lactate production and efflux over 40 min of exercise. The decreased rate of glycogen breakdown in hyperoxia was related to a decreased epinephrine concentration and an attenuated accumulation of potent allosteric effectors ADP_f and AMP_f during exercise, probably caused by a greater phosphorylation potential due to a significantly diminished rate of PCr utilization during hyperoxia.