The proglucagon gene (glu) encodes the incretin hormone GLP-1, produced in the intestinal endocrine L cells. We found previously that the bipartite transcription factor -catenin/TCF (cat/TCF), the major effector of the canonical Wnt signaling pathway, activates intestinal glu expression and GLP-1 production. We show here that 100 nM insulin stimulated glu expression and enhanced GLP-1 content in the intestinal GLUTag L cell line, as well as in primary fetal rat intestinal cell cultures. Increased intestinal glu mRNA expression and GLP-1 content were also observed in vivo, in hyperinsulinemic MKR mice. In the GLUTag cells, insulin-induced activation of glu expression occurred through the same TCF site that mediates cat/TCF activation. PI3K inhibition, but not PKB inhibition, attenuated the stimulation by insulin. Furthermore, nuclear -cat content in the intestinal L cells was increased by insulin. Finally, insulin enhanced the binding of TCF-4 and -cat to the TCF site in the glu promoter G2 enhancer element, determined by quantitative chromatin immunoprecipitation (ChIP) assay. Collectively, these findings indicate that enhancement of -cat nuclear translocation and cat/TCF binding are among the mechanisms underlying crosstalk between the insulin and Wnt signaling pathways in intestinal endocrine L cells.