β-catenin is a multifunctional protein involved in cell-cell adhesion and the Wnt signaling pathway. β-Catenin is activated upon its dephosphorylation, an event triggered by Dishevelled (Dvl)-mediated phosphorylation and deactivation of glycogen synthase kinase-3β (GSK-3β). In skeletal muscle, both insulin and exercise decrease GSK-3β activity, and we tested the hypothesis that these two stimuli regulate β-catenin. Immunoblotting demonstrated that Dvl, Axin, GSK-3β, and β-catenin proteins are expressed in rat red and white gastrocnemius muscles. Treadmill running exercise in vivo significantly decreased β-catenin phosphorylation in both muscle types, with complete dephosphorylation being elicited by maximal exercise. β-Catenin dephosphorylation was intensity dependent, as dephosphorylation was highly correlated with muscle glycogen depletion during exercise (r² = 0.84, P < 0.001). β-Catenin dephosphorylation was accompanied by increases in GSK-3β Ser⁹ phosphorylation and Dvl-GSK-3β association. In contrast to exercise, maximal insulin treatment (1 U/kg body wt) had no effect on skeletal muscle β-catenin phosphorylation or Dvl-GSK-3β interaction. In conclusion, exercise in vivo, but not insulin, increases the association between Dvl and GSK-3β in skeletal muscle, an event paralleled by β-catenin dephosphorylation.