The involvement of the β-isoform of glycogen synthase kinase (GSK-3) in glucose metabolism and insulin action was investigated in cultured human skeletal muscle cells. A 60% reduction in GSK-3β protein expression was attained by treatment with siRNA; GSK-3α expression was unaltered. GSK-3β knockdown did not influence total glycogen synthase (GS) activity, but increased the phosphorylation-dependent activity (fractional velocity–FV) in the basal state. Insulin responsiveness of GSFV was doubled by GSK-3β knockdown (p<0.05). Basal rates of glucose uptake (GU) were not significantly influenced by GSK-3β knockdown, while insulin stimulation of GU was increased. Improvements in insulin action on GS and GU did not involve changes in protein expression of either IRS-1 or Akt 1/2. Maximal insulin stimulation of phosphorylation of Akt was unaltered by GSK-3β knockdown. Unlike GSK-3α, GSK-3β directly regulates both GS activity in the absence of added insulin and through control of insulin action.