SOX9 inactivation is frequent in colorectal cancer (CRC) due to SOX9 gene mutations and/or to ectopic expression of MiniSOX9, a dominant negative inhibitor of SOX9. In the present study, we report a heterozygous L142P inactivating mutation of SOX9 in the DLD-1 CRC cell line and we demonstrate that the conditional expression of a wild type SOX9 in this cell line inhibits cell growth, clonal capacity and colonosphere formation while decreasing both the activity of the oncogenic Wnt/ß-catenin signaling pathway and the expression of the c-myc oncogene. This activity does not require SOX9 transcriptional function but, rather, involves an interaction of SOX9 with nuclear ß-catenin. Furthermore, we report that SOX9 inhibits tumor development when conditionally expressed in CRC cells injected either subcutaneous or intraperitoneous in BALB/c mice as an abdominal metastasis model. These observations argue in favor of a tumor suppressor activity for SOX9. As an siRNA targeting SOX9 paradoxically also inhibits DLD-1 and HCT116 CRC cell growth, we conclude that there is a critical level of endogenous active SOX9 needed to maintain CRC cell growth.