BACKGROUND & AIMS: In the normal intestinal epithelium, SOX9 expression is localized in the crypt cells. SOX9 is also expressed in diverse cancers, including colorectal cancer (CRC). Our previous loss-of-function study showed increased proliferation in Sox9-deficient crypts, indicating SOX9 suppresses cellular proliferation. We examined role of SOX9 in proliferation of normal intestinal epithelium, Apcmin/+ mouse adenomas, and CRC cells. METHODS: Sox9 deficient Apcmin/+mice were generated to investigate role of SOX9 in tumorigenesis. Crypt epithelial cells isolated from Sox9-deficient mice were also used to identify the potential target genes of SOX9. RESULTS: Sox9 deficiency in Apcmin/+ mice resulted in increased tumor burden relative to Apcmin/+ control mice. Insulin-like growth factor-binding protein 4 (IGFBP-4), a well documented inhibitor of the IGF/IGFR axis, was significantly downregulated in Sox9-deficient intestinal epithelial cells as well as in adenoma cells of Sox9-deficient Apcmin/+ mice. Co-staining experiments revealed colocalization of IGFBP-4 and SOX9 in mouse and human intestinal epithelial cells as well as primary CRC specimens. Reporter assays and chromatin immunoprecipitation (ChIP) demonstrated direct binding of SOX9 to IGFBP-4 promoter. Overexpression of SOX9 attenuated cellular proliferation, which was restored following treatment with a neutralizing antibody against IGFBP4, suggesting that SOX9 requires IGFBP-4 to suppress cellular proliferation. CONCLUSION: SOX9 suppresses cellular proliferation in intestinal epithelium, adenoma cells of Apcmin/+ mice, and CRC cells through activation of IGFBP-4.