Not only chronic exercise training, but also a single bout of exercise, increase insulin-stimulated glucose uptake in skeletal muscle. However, it is not well described how adaptations to exercise training affect the ability of a single bout of exercise to increase insulin sensitivity. Rodent studies suggest that the insulin-sensitizing effect of a single bout of exercise is AMPK-dependent (presumably via the α₂ β₂ γ₃ AMPK complex). Whether this is also the case in humans is unknown. Previous studies have shown that exercise training decreases expression of the α₂ β₂ γ₃ AMPK complex and diminishes the activation of this complex during exercise. Thus, we hypothesized that exercise training diminishes the ability of a single bout of exercise to enhance muscle insulin sensitivity. We investigated nine, healthy male subjects who performed one-legged knee-extensor exercise at the same relative intensity before and after 12 weeks of exercise training. Training increased VO_2peak and expression of mitochondrial proteins in muscle, while expression of AMPKγ3 was decreased. Training also increased whole body as well as muscle insulin sensitivity. Interestingly, insulin-stimulated glucose uptake in the acutely exercised leg was not further enhanced by training. Thus, the increase in insulin-stimulated glucose uptake following a single bout of one-legged exercise was lower in the trained vs. untrained state. This was associated with reduced signalling through confirmed α₂ β₂ γ₃ AMPK downstream targets (ACC and TBC1D4). These results suggest that the insulin-sensitizing effect of a single bout of exercise is also AMPK-dependent in human skeletal muscle.