Hepatocyte nuclear factor-4alpha (HNF-4alpha) serves as target for fatty acid nutrients and xenobiotic amphipathic carboxylates and may account for the differential effects of dietary fatty acids on colorectal cancer (CRC). The putative role played by HNF-4alpha in CRC has been verified here by evaluating the effect of HNF-4alpha antagonists and HNF-4alpha siRNA on CRC growth and proliferation in cultured CRC cells and xenotransplanted nude mice in vivo. HNF-4alpha ligand antagonists of the MEDICA series, namely, beta,beta'-tetramethylhexadecanedioic acid (M16betabeta) and gamma,gamma'-tetramethyloctadocanedioic acid (M18gammagamma) as well as HNF-4alpha siRNA are shown here to inhibit growth and proliferation of HT29 and Caco2 CRC cells, accompanied by increased subG1 cell population, downregulated PCNA, activation of caspase-3, upregulation of Bak and cytoplasmic cytochrome-c, and downregulation of Bcl-2 resulting in apoptotic death. Inhibition of CRC growth with concomitant apoptosis was further confirmed in nude mice xenotransplanted with HT29 CRC cells. CRC suppression by HNF-4alpha ligand antagonists and by HNF-4alpha siRNA was accounted for by suppression of HNF-4alpha transcription and protein expression. alpha,alpha'-tetrachlorotetradecanedioic acid (Cl-DICA), a MEDICA analogue that fails to suppress HNF-4alpha, was ineffective in suppressing growth of cultured or xenotransplanted HT29 CRC cells. Hence, increased transcriptional activity of HNF-4alpha converging onto genes coding for antiapoptotic oncogenes and cytokines may promote CRC development. Suppression of HNF-4alpha activity by natural or xenobiotic HNF-4alpha ligand antagonists or by HNF-4alpha siRNA may offer a treatment mode for CRC.