Beta-catenin is a multifunctional protein serving both as a structural element in cell adhesion and as a signaling component in the Wnt pathway, regulating embryogenesis and tumorigenesis. The signaling fraction of beta-catenin is tightly controlled by the adenomatous polyposis coli-axin-glycogen synthase kinase 3beta complex, which targets it for proteasomal degradation. It has been recently shown that Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Here we have highlighted the critical relevance of constitutive calpain pathway in the control of beta-catenin levels and functions, showing that small interference RNA knock down of endogenous calpain per se (i.e. in the absence of external stimuli) induces an increase in the free transcriptional competent pool of endogenous beta-catenin. We further characterized the role of the known calpain inhibitors, Gas2 and Calpastatin, demonstrating that they can also control levels, function, and localization of beta-catenin through endogenous calpain regulation. Finally we present Gas2 dominant negative (Gas2DN) as a new tool for regulating calpain activity, providing evidence that it counteracts the described effects of both Gas2 and Calpastatin on beta-catenin and that it works via calpain independently of the classical glycogen synthase kinase 3beta and proteasome pathway. Moreover, we provide in vitro biochemical evidence showing that Gas2DN can increase the activity of calpain and that in vivo it can induce degradation of stabilized/mutated beta-catenin. In fact, in a context where the classical proteasome pathway is impaired, as in colon cancer cells, Gas2DN biological effects accounted for a significant reduction in proliferation and anchorage-independent growth of colon cancer.