AS160 is an Akt substrate implicated in the regulation of both insulin and contraction mediated GLUT4 translocation and glucose uptake. The effect of aerobic exercise and subsequent insulin stimulation on AS160 phosphorylation and the binding capacity of 14-3-3, a novel protein involved in the dissociation of AS160 from GLUT4 vesicles, in human skeletal muscle are unknown. Hyperinsulinemic euglycemic clamps were performed on 7 males at rest, and immediately and 3 hr following a single bout of cycling exercise. Skeletal muscle biopsies were taken before and after the clamps. The insulin sensitivity index calculated during the final 30 min of the clamp was 8.0 ± 0.8, 9.1 ± 0.5 and 9.2 ± 0.8 for the Rest, Post-Ex and 3hr-Post-Ex trials, respectively. AS160 phosphorylation increased immediately following exercise and remained elevated 3 hr after exercise. In contrast, the 14-3-3 binding capacity of AS160, and phosphorylation of Akt and AMPK were only increased immediately following exercise. Insulin increased AS160 phosphorylation and 14-3-3 binding capacity, and IRS-1 and Akt phosphorylation, but the response to insulin was not enhanced by prior exercise. In conclusion, the 14-3-3 binding capacity of AS160 is increased immediately following acute exercise in human skeletal muscle, but this is not maintained 3 hr after exercise completion despite sustained AS160 phosphorylation. Insulin increases AS160 phosphorylation and 14-3-3 binding capacity, but prior exercise does not appear to enhance the response to insulin.