Because greater Akt substrate of 160 kDa (AS160) phosphorylation has been reported in insulin-stimulated skeletal muscles without improved Akt activation several hours post-exercise, we hypothesized that prior exercise would result in attenuated AS160 dephosphorylation in insulin-stimulated rat skeletal muscle. Epitrochlearis muscles were isolated from rats that were sedentary (SED) or exercised 3 h earlier (3 h post-exercise; 3hPEX). Paired muscles were incubated with [(3)H]-2-deoxyglucose (2-DG) without insulin or with insulin. Lysates from other insulin-stimulated muscles from SED or 3hPEX rats were evaluated using AS160(Thr642) and AS160(Ser588) dephosphorylation assays. Prior exercise led to greater 2-DG uptake concomitant with greater AS160(Thr642) phosphorylation and a non-significant trend (P=0.087) for greater AS160(Ser588). Prior exercise also reduced AS160(Thr642) and AS160(Ser588) dephosphorylation rates. These results support the idea that attenuated AS160 dephosphorylation may favor greater AS160 phosphorylation post-exercise.