Wnt/beta-catenin signaling is critical for a variety of fundamental cellular processes. Here, we investigated the implication of the Wnt/beta-catenin signaling in the in vivo regulation of beta-cell growth and regeneration in normal and diabetic rats. To this aim, TCF7L2, the distal effector of the canonical Wnt pathway, was knocked down in groups of normal and diabetic rats by the use of specific antisense morpholino-oligonucleotides. In other groups of diabetic rats, the Wnt/beta-catenin pathway was activated by the inhibition of its negative regulator GSK-3beta. GSK-3beta was inactivated by either LiCl or anti-GSK-3beta oligonucleotides. The beta-cell mass was evaluated by morphometry. beta-cell proliferation was assessed in vivo and in vitro by BrdU incorporation method. In vivo beta-cell neogenesis was estimated by the evaluation of PDX1-positive ductal cells and GLUT2-positive ductal cells and the number of beta cells budding from the ducts. We showed that the in vivo disruption of the canonical Wnt pathway resulted in the alteration of normal and compensatory growth of beta-cells mainly through the inhibition of beta-cell proliferation. Conversely, activation of the Wnt pathway through the inhibition of GSK-3beta had a significant stimulatory effect on beta-cell regeneration in diabetic rats. In vitro, GSK-3beta inactivation resulted in the stimulation of beta-cell proliferation. This was mediated by the stabilization of beta-catenin and the induction of cyclin D. Taken together, our results demonstrate the involvement of the canonical Wnt signaling in the neonatal regulation of normal and regenerative growth of pancreatic beta-cells. Moreover, we provide evidence that activation of this pathway by pharmacological maneuvers can efficiently improve beta-cell regeneration in diabetic rats. These findings might have potential clinical applications in the regenerative therapy of diabetes.