Colorectal cancers (CRCs) are classified as having microsatellite instability (MSI) or chromosomal instability (CIN); herein termed microsatellite stable (MSS). MSI colon cancers frequently display a poorly differentiated histology for which the molecular basis is not well understood. Gene expression and immunohistochemical profiling of MSS and MSI CRC cell lines and tumors revealed significant down-regulation of the intestinal-specific cytoskeletal protein villin in MSI colon cancer, with complete absence in 62% and 17% of MSI cell lines and tumors, respectively. Investigation of 577 CRCs linked loss of villin expression to poorly differentiated histology in MSI and MSS tumors. Furthermore, mislocalization of villin from the membrane was prognostic for poorer outcome in MSS patients. Loss of villin expression was not due to coding sequence mutations, epigenetic inactivation, or promoter mutation. Conversely, in transient transfection assays villin promoter activity reflected endogenous villin expression, suggesting transcriptional control. A screen of gut-specific transcription factors revealed a significant correlation between expression of villin and the homeobox transcription factor Cdx-1. Cdx-1 overexpression induced villin promoter activity, Cdx-1 knockdown down-regulated endogenous villin expression, and deletion of a key Cdx-binding site within the villin promoter attenuated promoter activity. Loss of Cdx-1 expression in CRC lines was associated with Cdx-1 promoter methylation. These findings demonstrate that loss of villin expression due to Cdx-1 loss is a feature of poorly differentiated CRCs. © 2012 American Society for Investigative Pathology.