An association between glycogen synthase kinase-3 (GSK3) in skeletal muscle and insulin resistance has been demonstrated in type 2 diabetic patients. In addition, inhibition of GSK3 improves insulin action. The aim of the present study was to elucidate the role of the - isoform of GSK3 in insulin resistance in human skeletal muscle cells from non-diabetic subjects maintained in culture. Transfection of muscle cells with specific antisense oligonucleotides resulted in a 30-50% decrease of GSK3 protein expression (p<0.05). While neither the basal fractional velocity of glycogen synthase (GSFV) (an indicator of the activation state of the enzyme) nor glucose uptake (GU) were altered, reducing GSK3 expression resulted in increases in insulin stimulation of both GS FV and GU. GSK3 over-expression (60-100% increase over control) did not alter basal GS FV or GU, but impaired insulin stimulation of both responses. Knockdown of GSK also led to an increase in IRS-1 protein expression while not altering insulin stimulation of pS473-Akt phosphorylation. However, GSK3 overexpression impaired insulin action on pS473-Akt. In summary, 1) Modulation of GSK3 expression has no effect on basal GU and GS activities. 2) Reduction of GSK3 expression results in improvements in insulin action. 3) Elevation of GSK3 in human skeletal muscle cells can induce insulin resistance for several responses. We conclude that GSK3 is an important regulator of muscle insulin action.