Glycogen synthase (GS) is the rate-limiting enzyme controlling nonoxidative glucose disposal in skeletal muscle. A reduction in GS activity and an impaired insulin responsiveness are characteristic features of skeletal muscle in type 2 diabetes. These properties also exist in human skeletal muscle cell cultures from type 2 diabetic subjects. To determine the effect of an isolated reduction in GS on skeletal muscle insulin action, cultures from nondiabetic subjects were treated with antisense oligonucleotides (ODNs) to GS to interfere with expression of the gene. Treatment with antisense ODNs reduced GS protein expression by 70% compared with control (scrambled) ODNs (P < .01). GS activity measured at 0.01 mmol/L glucose-6-phosphate (G-6-P) was reduced by antisense ODN treatment. The insulin responsiveness of GS was impaired. Insulin also failed to stimulate glucose incorporation into glycogen after antisense ODN treatment. The cellular glycogen content was lower in antisense ODN-treated cells compared with control ODN. The insulin responsiveness of glucose uptake was abolished by antisense ODN treatment. Thus, reductions in GS expression in human skeletal muscle cells lead to impairments in insulin responsiveness and may play an important role in insulin-resistant states.