Transmembrane prostate androgen-induced protein (TMEPAI) is a type I transmembrane protein induced by several intracellular signaling pathways such as androgen, TGF-β, EGF, and Wnt signaling. It has been reported that TMEPAI functions to suppress TGF-β and androgen signaling but here, we report a novel function of TMEPAI in Wnt signaling suppression. First, we show that TMEPAI significantly inhibits TCF/LEF transcriptional activity stimulated by Wnt3A, LiCl, and β-catenin. Mechanistically, TMEPAI overexpression prevented β-catenin accumulation in the nucleus and TMEPAI knockout in triple negative breast cancer cell lines promoted β-catenin stability and nuclear accumulation together with increased mRNA levels of Wnt target genes AXIN2 and c-MYC. The presence of TGF-β type I receptor kinase inhibitor did not affect the enhanced mRNA expression of AXIN2 in TMEPAI knockout cells. These data suggest that TMEPAI suppresses Wnt signaling by interfering with β-catenin stability and nuclear translocation in a TGF-β signaling-independent manner.