CDX2 is a Drosophila caudal-related homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. We have reported that CDX2 promotes tumorigenicity in a subset of human colorectal cancer cell lines. Here, we present evidence that CDX2 negatively regulates the well-documented growth inhibitor insulin-like growth factor binding protein-3 (IGFBP-3). Specifically, CDX2 binds to the IGFBP-3 gene promoter and can repress IGFBP-3 transcription, protein expression and secretion. Furthermore, inhibition of IGFBP-3 partially rescues the decreased anchorage-independent growth phenotype observed in CDX2 knockout cells. These data demonstrate for the first time that (1) CDX2 can function as a transcriptional repressor, and (2) one mechanism by which CDX2 promotes anchorage-independent growth is by transcriptional repression of IGFBP-3.