Hepatocyte nuclear factor (HNF) 4alpha is a liver-enriched nuclear receptor that plays a critical role in regulating the expression of numerous hepatic genes, including members of the cytochrome P450 (CYP) superfamily, several of which are expressed in a sex-dependent manner. Presently, we use a liver-specific Hnf4alpha-deficient mouse model to investigate the role of HNF4alpha in regulating liver-enriched transcription factors and sexually dimorphic Cyps in liver in vivo. Real-time PCR analysis of RNA isolated from livers of wild-type and Hnf4alpha-deficient mice revealed the following: 1) HNF4alpha exerts both positive regulation (Hnfalpha, C/ebpalpha, and C/ebpbeta) and negative regulation (Hnf3alpha and the HNF4alpha coactivator Pgc-1alpha) on liver transcription factor expression; 2) a strong dependence on HNF4alpha characterizes several male-predominant Cyps (2d9 and 8b1), female-predominant Cyps (2b10, 2b13, 3a41, and 3a44) and Cyps, whose expression is sex independent (3a11, 3a25); 3) HNF4alpha confers a unique, positive regulation of two male-expressed genes (Cyp4a12 and GSTpi) and a negative regulation of several female-predominant genes (Cyp2a4, Cyp2b9, Hnf3beta, and Hnf6), both of which are manifest in male but not female mouse liver. These trends were confirmed at the protein level by Western blot analysis using antibodies raised to Cyp2a, Cyp2b, and Cyp3a family members. Thus, HNF4alpha is an essential player in the complex regulatory network of liver-enriched transcription factors and the sexually dimorphic mouse Cyp genes that they regulate. HNF4alpha is proposed to contribute to the sex specificity of liver gene expression by positively regulating a subset of male-specific Cyp genes while concomitantly inhibiting the expression of certain female-specific Cyps and liver transcription factors, by mechanisms that are operative in male, but not female, mouse liver.