The POZ-zinc finger transcription factor Kaiso was first identified as a specific binding partner for the Armadillo catenin and cell adhesion cofactor, p120^ctn. Kaiso is a unique POZ protein with bi-modal DNA-binding properties; it associates with a sequence-specific DNA consensus Kaiso binding site (KBS) or methylated CpG dinucleotides, and regulates transcription of artificial promoters containing either site. Interestingly, the promoter of the Wnt/β-catenin/TCF target gene matrilysin possesses two conserved copies of the KBS, which suggested that Kaiso might regulate matrilysin expression. In this study, we demonstrate using chromatin immunoprecipitation analysis that Kaiso associates with the matrilysin promoter in vivo. Minimal promoter assays further confirmed that Kaiso specifically repressed transcription of the matrilysin promoter; mutation of the KBS element or RNAi-mediated depletion of Kaiso abrogated this effect. More importantly, Kaiso blocked β-catenin-mediated activation of the matrilysin promoter. Consistent with our previous findings, both Kaiso-DNA binding and Kaiso-mediated transcriptional repression of the matrilysin promoter were inhibited by overexpression of wild-type p120^ctn, but not by a p120^ctn mutant exhibiting impaired nuclear import. Collectively, our data establish Kaiso as a sequence-specific transcriptional repressor of the matrilysin promoter, and suggest that p120^ctn and β-catenin act in a synergistic manner, via distinct mechanisms, to activate matrilysin expression.