Beta-catenin (-catenin) is a multifunctional protein involved in cell-cell adhesion and the Wnt signaling pathway. -catenin is activated upon its dephosphorylation, an event triggered by dishevelled (Dvl)-mediated phosphorylation and deactivation of glycogen synthase kinase-3 (GSK-3). In skeletal muscle, both insulin and exercise decrease GSK-3 activity and we tested the hypothesis that these two stimuli regulate -catenin. Immunoblotting demonstrated that Dvl, Axin, GSK-3 and -catenin proteins are expressed in rat red and white gastrocnemius muscles. Treadmill running exercise in vivo significantly decreased -catenin phosphorylation in both muscle types, with complete dephosphorylation being elicited by maximal exercise. -catenin dephosphorylation was intensity dependent, as dephosphorylation was highly correlated with muscle glycogen depletion during exercise (R²=0.84, P<0.001). -catenin dephosphorylation was accompanied by increases in GSK-3 Ser⁹ phosphorylation and Dvl- GSK-3 association. In contrast to exercise, maximal insulin treatment (0.1U/kg body weight) had no effect on skeletal muscle -catenin phosphorylation or Dvl-GSK-3 interaction. In conclusion, exercise in vivo, but not insulin, increases the association between Dvl and GSK-3 in skeletal muscle, an event paralleled by -catenin dephosphorylation.