Dysregulation of Wnt/β-catenin pathway plays a central role in early events in colorectal carcinogenesis. We examined the effect of quercetin, a famous anti-tumor agent, against β-catenin/Tcf signaling in SW480 cells. Quercetin inhibited the transcriptional activity of β-catenin/Tcf in SW480 and also in HEK293 cells transiently transfected with constitutively active mutant β-catenin gene, whose product is not induced to be degraded by APC–Axin–GSK3β complex, so we concluded that its inhibitory mechanism was related to β-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed EMSA showing that binding of the Tcf complexes to its specific DNA-binding sites was strongly suppressed by quercetin. Immunoprecipitation analysis also showed that the binding of β-catenin to Tcf-4 was also disrupted by quercetin. Western blot analysis proved these decreased bindings resulted from decreased level of β-catenin and Tcf-4 product in nucleus caused by quercetin. Together, we suggest that quercetin is an excellent inhibitor of β-catenin/Tcf signaling in SW480 cell lines, and the reduced β-catenin/Tcf transcriptional activity is due to the decreased nuclear β-catenin and Tcf-4 proteins.