This study evaluated if improved insulin stimulated glucose uptake in recovery from acute exercise coincided with reduced malonyl-CoA (MCoA) content in human muscle. Furthermore, we investigated if a high fat diet (65 energy-% (FAT)) would alter the content of MCoA and insulin action when compared to a high carbohydrate diet (65 energy-% (CHO)). After 4 days of iso-caloric diet on two occasions (FAT/CHO), 12 male subjects performed 1 hour of one-legged knee-extensor exercise (~80% peak work load). Four hours after exercise, insulin stimulated glucose uptake was determined in both legs during a euglycemic-hyperinsulinemic clamp. Muscle biopsies were obtained in both legs before and after the clamp. Four hours after exercise, insulin stimulated glucose uptake was improved (~70%,P<0.001) independent of diet composition and despite normal insulin stimulated regulation of IRS-1 associated PI-3 kinase, Akt, GSK-3 and GS. Interestingly, exercise resulted in a sustained reduction (~20%,P<0.05) in MCoA content 4 hours after exercise that correlated (r=0.65,P<0.001) with improved insulin stimulated glucose uptake. Four days of FAT diet resulted in an increased content of IMTG (P<0.01) but did not influence muscle MCoA content or whole body insulin stimulated glucose uptake. However, at the muscular level proximal insulin signaling and insulin stimulated glucose uptake appeared to be compromised although to a minor extent by the FAT diet. Collectively, this study indicates that reduced muscle MCoA content in recovery from exercise may be part of the adaptive response leading to improved insulin action on glucose uptake after exercise in human muscle.