Following injury, a large number of hepatic acute phase genes are rapidly modulated at the transcriptional level to restore metabolic homeostasis and limit tissue damage. Hepatocyte nuclear factor 4alpha (HNF-4alpha) is a liver-enriched transcription factor that controls embryonic liver development and regulates tissue-specific gene expression in adult liver cells. Many genes encoding acute phase proteins contain HNF-4alpha-binding sites in their promoter regions and are transcriptionally regulated by HNF-4alpha. Utilizing a cytokine induced acute phase response in HepG2 cells, we investigated the role of HNF-4alpha in regulating the transcription of three HNF-4alpha sensitive genes, alpha1-antitrypsin (alpha1-AT), transthyretin (TTR), and apolipoprotein B (ApoB) after injury. The transcriptional behavior of all three genes depends, in part, on the intracellular concentrations of HNF-4alpha. However, the unique mRNA expression patterns of alpha1-AT, TTR, and ApoB in response to cytokine treatment were abrogated in HepG2 cells with dramatically reduced HNF-4alpha protein concentrations. The mechanism by which HNF-4alpha mediates this injury response is through site-specific alterations in HNF-4alpha-binding abilities and transactivation potentials. Cytokine treatment phosphorylates HNF-4alpha, which directly affects HNF-4alpha activity. Our results demonstrate that HNF-4alpha is a crucial mediator in the regulation of alpha1-AT, TTR, and ApoB gene expression before and after injury, providing evidence of a novel role for HNF-4alpha in the control of the liver's acute phase response.