Background: Phosphorylation of AS160 and TBC1D1 plays an important role for GLUT4 mobilization to the cell surface. The phosphorylation of AS160 and TBC1D1 in humans in response to acute exercise is not fully characterized. Objective: To study AS160 and TBC1D1 phosphorylation in human skeletal muscle after aerobic exercise followed by a hyperinsulinemic euglycemic clamp. Design: Eight healthy males were studied on two occasions: 1) in the resting state and 2) in the hours after a one-hour bout of ergometer-cycling. A hyperinsulinemic euglycemic clamp was initiated 240 minutes after exercise and in a time-matched non-exercised control condition. We obtained muscle biopsies 30 minutes after exercise and in a time-matched non-exercised control condition (t=30) and after 30 minutes of insulin stimulation (t=270) and investigated site-specific phosphorylation of AS160 and TBC1D1. Results: Phosphorylation on AS160 and TBC1D1 was increased 30 minutes after the exercise bout while phosphorylation of the putative upstream kinases, Akt and AMPK, was unchanged compared to resting control condition. Exercise augmented insulin-stimulated phosphorylation on AS160 at Ser(341) and Ser(704) 270 minutes after exercise. No additional exercise effects were observed on insulin-stimulated phosphorylation of Thr(642) and Ser(588) on AS160 or Ser(237) and Thr(596) on TBC1D1. Conclusions: AS160 and TBC1D1 phosphorylations were evident 30 minutes after exercise without simultaneously increased Akt and AMPK phosphorylation. Unlike TBC1D1, insulin-stimulated site-specific AS160 phosphorylation is modified by prior exercise, but these sites do not include Thr(642) and Ser(588). Together, these data provide new insights into phosphorylation of key regulators of glucose transport in human skeletal muscle.