Insulin stimulates glycogen synthase (GS) through dephosphorylation of serine residues, and this effect is impaired in skeletal muscle from insulin-resistant [obese and type 2 diabetic (T2DM)] subjects. Exercise also increases GS activity, yet it is not known whether the ability of exercise to affect GS is impaired in insulin-resistant subjects. The objective of this study was to examine the effect of acute exercise on GS phosphorylation and enzyme kinetic properties in muscle from insulin-resistant individuals. Lean normal glucose-tolerant (NGT), obese NGT, and obese T2DM subjects performed 40 min of moderate-intensity cycle exercise (70% of Vo(2max)). GS kinetic properties and phosphorylation were measured in vastus lateralis muscle before exercise, immediately after exercise, and 3.5 h postexercise. In lean subjects, GS fractional activity increased twofold after 40 min of exercise, and it remained elevated after the 3.5-h rest period. Importantly, exercise also decreased GS K(m) for UDP-glucose from ≈0.5 to ≈0.2 mM. In lean subjects, exercise caused significant dephosphorylation of GS by 50-70% (Ser(641), Ser(645), and Ser(645,649,653,657)), and phosphorylation of these sites remained decreased after 3.5 h; Ser⁷ phosphorylation was not regulated by exercise. In obese NGT and T2DM subjects, exercise increased GS fractional activity, decreased K(m) for UDP-glucose, and decreased GS phosphorylation as effectively as in lean NGT subjects. We conclude that the molecular regulatory process by which exercise promotes glycogen synthesis in muscle is preserved in insulin-resistant subjects.