The structural maintenance of chromosome protein SMC3 is a component of the cohesin complex that mediates sister chromatid cohesion and segregation in prokaryotes and eukaryotes. It is also present extracellularly in the form of a chondroitin sulfate proteoglycan known as bamacan. We have found previously that SMC3 expression is elevated in a large fraction of human colon carcinomas. The additional finding that the protein is significantly increased in the intestinal polyps of Apc Min/؉ mice has led us to hypothesize that SMC3 expression is linked to activation of the APC/␤-catenin/ TCF4 pathway. The immunohistochemical analysis of colon adenocarcinomas from clinical specimens revealed that ␤-catenin and SMC3 antigens co-localize with maximal stain intensity within the transformed areas. Cloning and sequencing of 1578 bp of the human SMC3 promoter unveiled the presence of seven putative consensus sequences for ␤-catenin/TCF4 binding, two of which are conserved in the mouse Smc3 promoter. Transient transfection experiments in HCT116 and SW480 human colon carcinoma cells using deletion and mutated promoter constructs in luciferase reporter vectors confirmed that the putative sites, the first located at ؊48 bp and the second located at ؊701 bp, are susceptible to ␤-catenin/TCF4 transactivation. Co-transfection with a ␤-catenin expression vector enhanced the promoter activity whereas E-cadherin had the opposite effect. Binding of ␤-catenin/TCF4 complexes from SW480 nuclear extracts to these sequences was confirmed by electrophoretic shift and supershift mobility assays. Altogether these results are consistent with the idea that the ␤-catenin/TCF4 transactivation pathway contributes to SMC3 overexpression in intestinal tumorigenesis.