Hepatocyte nuclear factor (HNF) 4 is a liver-enriched transcription factor that regulates numerous liver-expressed genes including several sex-specific cytochrome P450 genes. Presently, a liver-specific HNF4-deficient mouse model was used to characterize the impact of liver HNF4 deficiency on a global scale using 41,174 feature microarrays. A total of 4994 HNF4-dependent genes were identified, of which 1000 fewer genes responded to the loss of HNF4 in female liver as compared to male liver. Sex differences in the impact of liver HNF4 deficiency were even more dramatic when genes showing sex-specific expression were examined. Thus, 372 of the 646 sex-specific genes characterized by a dependence on HNF4 responded to the loss of HNF4 in males only, as compared to only 61 genes that responded in females only. Moreover, in male liver, 78% of 508 male-specific genes were down-regulated and 42% of 356 female-specific genes were up-regulated in response to the loss of HNF4, with sex specificity lost for 90% of sex-specific genes. This response to HNF4 deficiency is similar to the response of male mice deficient in the GH-activated transcription factor STAT5b, where 90% of male-specific genes were down-regulated and 61% of female-specific genes were up-regulated, suggesting these two factors cooperatively regulate liver sex-specificity by mechanisms that are primarily active in males. Finally, 203 of 648 genes previously shown to bind HNF4 in mouse hepatocytes were affected by HNF4 deficiency in mouse liver, with the HNF4-bound gene set 5-fold enriched for genes positively regulated by HNF4. Thus, a substantial fraction of the HNF4-dependent genes reported here are likely to be direct targets of HNF4.