Background: CDX2 plays a key part in the differentiation of Caco-2 cells, a colon carcinoma derived cell line that undergoes spontaneous differentiation. The effect of CDX2 expression in Caco-2 cells over time in culture has not been studied yet on a genome-wide level. Methods: The impact of CDX2 expression on the genomic profile of Caco-2 cells was studied by transducing cells with CDX2 targeting shRNAs. Knockdown efficiency was assessed on mRNA level and protein level by RTPCR, microarrays, and Western blots. Gene set enrichment analysis was performed to assess regulation of specific gene sets. Results: CDX2 expression had an inhibitory effect on the transcriptional activity of beta-catenin/TCF at early stages of culturing, while at later stages, its role in the trans-activation of target genes specific for small intestinal enterocytes seemed more dominant. Conclusions: The unique induction of a small intestinal signature upon differentiation in Caco-2 cells seems to be at least partially under the control of CDX2. (c) 2013 Elsevier B.V. All rights reserved.