Wnt/beta-catenin signaling plays an essential role in colon carcinogenesis. Galectin-3, a beta-galactoside-binding protein, has been implicated in Wnt signaling, but the precise mechanisms by which galectin-3 modulates the Wnt pathway are unknown. In the present study, we determined the effects of galectin-3 on the Wnt/beta-catenin pathway in colon cancer cells, as well as the mechanisms involved. Galectin-3 levels were manipulated in human colon cancer cells by stable transfection of galectin-3 antisense, short hairpin RNA, or full-length galectin-3 cDNA, and effects on beta-catenin levels, subcellular distribution, and Wnt signaling were determined. Galectin-3 levels correlated with beta-catenin levels in a variety of colon cancer cell lines. Down-regulation of galectin-3 resulted in decreased beta-catenin protein levels but no change in beta-catenin mRNA levels, suggesting that galectin-3 modulates beta-catenin by another mechanism. Reduction of galectin-3 led to reduced nuclear beta-catenin with a concomitant decrease in TCF4 transcriptional activity and expression of its target genes. Conversely, transfection of galectin-3 cDNA into colon cancer cells increased beta-catenin expression and TCF4 transcriptional activity. Down-regulation of galectin-3 resulted in AKT and glycogen synthase kinase-3beta (GSK-3beta) dephosphorylation and increased GSK activity, increasing beta-catenin phosphorylation and degradation. Ly294002, an inhibitor of phosphatidylinositol 3-kinase, and dominant-negative AKT, suppressed TCF4 transcriptional activity induced by galectin-3 whereas LiCl, a GSK-3beta inhibitor, increased TCF4 activity, mimicking the effects of galectin-3. These results suggest that galectin-3 mediates Wnt signaling, at least in part, by regulating GSK-3beta phosphorylation and activity via the phosphatidylinositol 3-kinase/AKT pathway, and, thus, the degradation of beta-catenin in colon cancer cells.