Insulin-stimulated muscle glucose uptake is inversely associated with the muscle glycogen concentration. To investigate whether this association is a cause and effect relationship, we compared insulin-stimulated muscle glucose uptake in non-contracted and post-contracted muscle of GSL3 transgenic and wildtype mice. GSL3 transgenic mice overexpress a constitutively active form of glycogen synthase, which results in an abundant storage of muscle glycogen. Muscle contraction was elicited by in situ electrical stimulation of the sciatic nerve. Right gastrocnemii from GSL3 transgenic and wildtype mice were subjected to 30 min of electrical stimulation followed by hindlimb perfusion of both hindlimbs. Thirty min of contraction significantly reduced muscle glycogen concentration in wildtype (49%) and transgenic (27%) mice although transgenic mice retained 168.8 ± 20.5 µmol ^. g^-1 glycogen compared to 17.7 ± 2.6 µmol ^. g^-1 glycogen for wildtype mice. Muscle of transgenic and wildtype mice demonstrated similar pre- (3.6 ± 0.3 µmol ^. g^{-1 .} h^-1 and 3.9 ± 0.6 µmol ^. g^{-1 .} h^-1, for transgenic and wildtype, respectively) and post-contraction (7.9 ± 0.4 µmol ^. g^{-1 .} h^-1 and 7.0 ± 0.4 µmol ^. g^{-1 .} h^-1, for transgenic and wildtype, respectively) insulin-stimulated glucose uptakes. However, the [¹⁴C] glucose incorporated into glycogen was greater in non-contracted (151%) and post-contracted (157%) transgenic muscle versus muscle of corresponding wildtype mice. These results indicate that glycogen synthase activity is not rate limiting for insulin-stimulated glucose uptake in skeletal muscle, and that the inverse relationship between muscle glycogen and insulin-stimulated glucose uptake is an association, not a cause and effect relationship.