The gut-specific homeotic transcription factor Cdx2 is a crucial regulator of intestinal development and homeostasis, which is downregulated in colorectal cancers (CRC) and exhibits a tumor suppressor function in the colon. We have previously established that several endodermal transcription factors, including HNF4 alpha and GATA6, are involved in Cdx2 regulation in the normal gut. Here we have studied the role of HNF4 alpha in the mechanism of deregulation of Cdx2 in colon cancers. Crossing Apc(Delta 14/+) mice prone to spontaneous intestinal tumor development with pCdx2-9LacZ transgenic mice containing the LacZ reporter under the control of the 9.3-kb Cdx2 promoter showed that this promoter segment contains sequences recapitulating the decrease of Cdx2 expression in intestinal cancers. Immunohistochemistry revealed that HNF4 alpha, unlike GATA6, exhibited a similar decrease to Cdx2 in genetic (Apc(min/+) and Apc(Delta 14/+)) and chemically induced (Azoxymethane (AOM) treatment) models of intestinal tumors in mice. HNF4 alpha and Cdx2 also exhibited a comparable deregulated pattern in human CRC. Correlated patterns were observed between HNF4 alpha and Cdx2 in several experimental models of human colon cancer cell lines: xenografts in nude mice, wound healing and glucose starvation. Furthermore, Cdx2 decreased by knocking down HNF4 alpha in human colon cancer cells using siRNA and in the colon of mice conditionally knocked out for the Hnf4 alpha gene in the adult intestine (Hnf4 alpha(f/f); VilCre(ERT2) mice). Finally, the conditionally knocked out mice Hnf4 alpha(f/f); VilCre(ERT2) treated with the carcinogen AOM developed colorectal tumors earlier than wild-type mice, as previously reported for mice with a reduced Cdx2 expression. In conclusion, this study provides evidence that the downregulation of HNF4 alpha is an important determinant of the reduced expression of the Cdx2 tumor suppressor gene in intestinal cancers. Consistently, similar to Cdx2, HNF4 alpha exerts a tumor suppressor function in the colon in that its loss of function facilitates tumor progression.