The functional consequences of up-regulation of β-catenin as a transcription factor are complex in different tumors. To clarify roles during squamous differentiation (SqD) of endometrial carcinoma (Em Ca) cells, we investigated expression of β-catenin, as well as cyclin D1, p53, p21WAF1, and PML (promyelocytic leukemia) in 80 cases of Em Ca with SqD areas, in comparison with cell proliferation determined with reference to Ki-67 antigen positivity. The impact of β-catenin-T-cell factor (TCF)-mediated transcription was also examined using Em Ca cells. In clinical cases, nuclear β-catenin accumulation was more frequent in SqD areas, being positively linked with expression of cyclin D1, p53, and p21WAF1, and inversely with Ki-67 and PML immunoreactivity. Significant correlations of nuclear β-catenin, cyclin D1, p53, and p21WAF1 were noted between SqD and the surrounding carcinoma lesions. The Ishikawa cell line, with stable or tetracycline-regulated expression of mutant β-catenin, showed an increase in expression levels of cyclin D1, p14ARF, p53, and p21WAF1 but not PML, and activation of β-catenin-TCF4-mediated transcription determined with TOP/FOP constructs. The cell morphology was senescence-like rather than squamoid in appearance. Moreover, overexpressed β-catenin could activate transcription from p14ARF and cyclin D1 promoters, in a TCF4-dependent manner. These findings indicate that in Em Cas, nuclear β-catenin can simultaneously induce activation of the p53-p21WAF1 pathway and overexpression of cyclin D1, leading to suppression of cell proliferation or induction of cell senescence. However, overexpression of β-catenin alone is not sufficient for development of a squamoid phenotype in Em Ca cells, suggesting that nuclear accumulation is an initial signal for trans-differentiation.