This is the first study to examine the effects of endurance training on the activation state of glycogen phosphorylase (Phos) and pyruvate dehydrogenase (PDH) in human skeletal muscle during exercise. We hypothesized that 7 wk of endurance training (Tr) would result in a posttransformationally regulated decrease in flux through Phos and an attenuated activation of PDH during exercise due to alterations in key allosteric modulators of these important enzymes. Eight healthy men (22 ± 1 yr) cycled to exhaustion at the same absolute workload (206 ± 5 W; ∼80% of initial maximal oxygen uptake) before and after Tr. Muscle biopsies (vastus lateralis) were obtained at rest and after 5 and 15 min of exercise. Fifteen minutes of exercise post-Tr resulted in an attenuated activation of PDH (pre-Tr: 3.75 ± 0.48 vs. post-Tr: 2.65 ± 0.38 mmol·min−1·kg wet wt−1), possibly due in part to lower pyruvate content (pre-Tr: 0.94 ± 0.14 vs. post-Tr: 0.46 ± 0.03 mmol/kg dry wt). The decreased pyruvate availability during exercise post-Tr may be due to a decreased muscle glycogenolytic rate (pre-Tr: 13.22 ± 1.01 vs. post-Tr: 7.36 ± 1.26 mmol·min−1·kg dry wt−1). Decreased glycogenolysis was likely mediated, in part, by posttransformational regulation of Phos, as evidenced by smaller net increases in calculated muscle free ADP (pre-Tr: 111 ± 16 vs. post-Tr: 84 ± 10 μmol/kg dry wt) and Pi(pre-Tr: 57.1 ± 7.9 vs. post-Tr: 28.6 ± 5.6 mmol/kg dry wt). We have demonstrated for the first time that several signals act to coordinately regulate Phos and PDH, and thus carbohydrate metabolism, in human skeletal muscle after 7 wk of endurance training.