Hepatocyte nuclear factor-4 (HNF-4) regulates transcription of several genes involved in lipid metabolism, including that of apolipoprotein (apo) A-IV, which is tightly regulated by lipid absorption and enhances enterocyte chylomicron secretion. Studies were performed to define the role of HNF-4 in the regulation of apo A-IV gene transcription by dietary fatty acid in neonatal swine small intestine. HNF-4 mRNA was expressed in liver > intestine > kidney in suckling, weanling and weaned pigs. Jejunal HNF-4 mRNA and protein and apo A-IV and MTP large subunit mRNA expression were induced in parallel in 2-day-old swine by a 24-hr high-fat intraduodenal infusion. In IPEC-1 cells, incubation with oleic acid (OA) resulted in coordinate induction of both HNF-4, apo A-IV, and MTP mRNA, similar to that observed in vivo. When HNF-4 expression was driven by doxycycline using the TET-On system in the absence of OA to observe the effect of HNF-4 directly on apo A-IV and MTP mRNA levels in the absence of other factors that might be concomitantly induced by fatty acid absorption, apo A-IV and MTP expression were increased. In luciferase reporter gene assays in IPEC-1 cells using apo A-IV/C-III intergenic region constructs, TET-On-regulated HNF-4 expression without OA increased luciferase activity, and incubation with OA did not further increase activity. These data suggest that acute induction of the apo A-IV and MTP genes by dietary lipid in newborn intestine occurs, at least in part, via ligand-independent transactivation by HNF-4 that is itself induced by a lipid-mediated mechanism.