BACKGROUND & AIMS: Wnt signaling contributes to the development of colorectal cancer (CRC). We studied interactions between lysine demethylase 4D (KDM4D or JMJD2D) and b-catenin, a mediator of Wnt signaling, in CRC cell lines and the effects on tumor formation in mice. METHODS: We obtained colorectal tumor specimens and surrounding nontumor colon tissues (controls) from patients undergoing surgery in China; levels of JMJD2D were measured by immunohistochemical or immunoblot analysis. JMJD2D expression was knocked down in CRC (CT26, HCT116, and SW480 cells) using small hairpin RNAs, and cells were analyzed with viability, flow cytometry, colony formation, and transwell migration and invasion assays. Cells were also grown as tumor xenografts in nude mice or injected into tail veins or spleens of mice, and metastases were measured. We performed promoter activity, co-immunoprecipitation, and chromatin immunoprecipitation assays. We also performed studies with Apcmin/thorn and JMJD2D-knockout mice; these mice were crossed, and colorectal tumor formation in offspring (Apcmin/thorn Jmjd2d thorn/thorn and Apcmin/thorn Jmjd2d-/-) was analyzed. JMJD2D-knockout and wild-type (control) mice were given azoxymethane followed by dextran sodium sulfate to induce colitis-associated CRC; some mice were given the JMJD2D inhibitor 5-chloro-8-hydroxyquinoline (5-c-8HQ) or vehicle to examine the effects of 5-c-8HQ on intestinal tumor formation. RESULTS: Levels of JMJD2D were significantly higher in human colorectal tumors than in control tissues and correlated with levels of proliferating cell nuclear antigen. JMJD2D knockdown reduced CRC cell proliferation, migration, and invasion, as well as growth of xenograft tumors and formation of metastases in mice. JMJD2D was required for expression of b-catenin in CRC cell lines; ectopic expression of JMJD2D increased the promoter activities of genes regulated by b-catenin (MYC, CCND1, MMP2, and MMP9). We found that JMJD2D and b-catenin interacted physically and that JMJD2D demethylated H3K9me3 at promoters of b-catenin target genes. JMJD2D-knockout mice developed fewer colitis-associated colorectal tumors than control mice, and their tumor tissues had lower levels of b-catenin, MYC, cyclin D1, and proliferating cell nuclear antigen than tumors from control mice. Apcmin/thorn Jmjd2d-/-mice developed fewer and smaller colon tumors than Apcmin/thorn mice. Mice given 5-c-8HQ developed smaller and fewer colitisassociated tumors, with lower levels of cell proliferation, than mice given vehicle. Apcmin/thorn mice given 5-c-8HQ also developed fewer tumors in intestines and colons than mice given vehicle. CONCLUSIONS: Levels of the histone demethylase JMJD2D are increased in human colorectal tumors compared with nontumor colon tissues. JMJD2D interacts with b-catenin to activate transcription of its target genes and promote CRC cell proliferation, migration, and invasion, as well as formation of colorectal tumors in mice.