Abstract Misregulation of the Wnt signaling pathway has been linked to many human cancers including colon carcinoma and melanoma. The primary mediator of the oncogenic effects of the Wnt signaling pathway is β-catenin. Accumulation of nuclear β-catenin and transcription activation of lymphoid enhancer factor 1 (LEF1)/T-cell factor (TCF) target genes underlie the oncogenic activity. However, the mechanism of β-catenin–mediated transcriptional activation remains poorly understood. In this study, we identified Mastermind-like 1 (Maml1), which is thought to be a specific coactivator for the Notch pathway, as a coactivator for β-catenin. We found that Maml1 participates in the Wnt signaling by modulating the β-catenin/TCF activity. We show in vivo that Maml1 is recruited by β-catenin on the cyclin D1 and c-Myc promoters. Importantly, we show that Maml1 functions in the Wnt/β-catenin pathway independently of Notch signaling. Finally, we show that the knockdown of Mastermind-like family proteins in colonic carcinoma cells results in cell death by affecting β-catenin–induced expression of cyclin D1 and c-Myc. This is the first demonstration of a role for the Mastermind-like family in another signaling pathway and that the knockdown of Mastermind-like family function leads to tumor cell death. [Cancer Res 2007;67(18):8690–8]