ABSTRACT The molecular causes leading to secondary liver malignancies are unknown. Here we report regulation of major hepatic nuclear factors in human colorectal liver metastases and primary colonic cancer. Notably, the genes coding for HNF6, HNF1β, and C/EBPγ were selectively regulated in liver metastases. We therefore studied protein expression of regulated transcription factors and found unacetylated HNF6 to be a hallmark of colorectal liver metastases. For its known interaction with HNF6, we investigated expression of FOXA2, which we found to be specifically induced in colorectal liver metastases. By electromobility shift assay, we examined DNA binding of disease regulated transcription factors. Essentially, no HNF6 DNA binding was observed. We also searched for sequence variations in the DNA binding domains of HNF6, but did not identify any mutation. Furthermore, we probed for expression of 28 genes targeted by HNF6. Mostly transcript expression was repressed except for tumor growth. In conclusion, we show HNF6 protein expression to be driven by the hepatic environment. Its expression is not observed in healthy colon or primary colonic cancer. HNF6 DNA binding is selectively abrogated through lack of post‐translational modification and interaction with FOXA2. Targeting of FOXA2 and HNF6 may therefore enable mechanism‐based therapy for colorectal liver metastases.—Lehner, F., Kulik, U., Klempnauer, J., Borlak, J. The hepatocyte nuclear factor 6 (HNF6) and FOXA2 are key regulators in colorectal liver metastases. FASEB J. 21, 1445–1462 (2007)