Intestinal microbiota influence diverse aspects of host nutrient metabolism and immunity in part by controlling tissue-specific transcription of key host genes. However, the host transcriptional regulatory mechanisms mediating microbial control of host gene expression are poorly understood. Microbiota colonization in zebrafish and mice leads to significant reductions in intestinal epithelial expression of Angiopotein-like protein 4 (angptl4/fiaf), a circulating inhibitor of lipoprotein lipase. We previously showed that a discrete DNA cis-regulatory module (CRM) at the zebrafish angptl4 locus named in3.4 mediates microbial control of transcription in the intestinal epithelium. To identify transcription factors that might regulate in3.4 activity, we used an unbiased yeast 1-hybrid approach and discovered that members of the Hepatic nuclear factor 4 (Hnf4) family of nuclear receptor transcription factors specifically bind to in3.4. Site-directed mutagenesis of in3.4 in yeast 1-hybrid and zebrafish transgenesis assays confirmed the existence of two Hnf4 binding sites in in3.4. Using the CRISPR/Cas9 system, we made targeted mutations in the zebrafish hnf4a gene and found that in3.4 enhancer activity was significantly attenuated in the intestinal epithelium of hnf4a homozygous mutants. To test the requirement for hnf4a on host transcriptional responses to microbiota, we used RNA-seq to compare transcript levels in the digestive tracts of hnf4a mutant and wild-type zebrafish raised germ-free or colonized with a conventional microbiota. Zebrafish hnf4a mutants displayed reduced expression of genes involved in lipid biosynthesis and metabolism, and increased expression of genes involved in inflammation and response to bacteria. Strikingly, loss of hnf4a significantly altered the expression of over half of the 822 genes that displayed differential expression in response to microbiota colonization. Hnf4a is an ancient member of the nuclear receptor family with important known roles in intestinal and hepatic physiology. Our results establish a novel role for Hnf4a in mediating host transcriptional responses to intestinal microbiota, and provide new mechanistic insight into how animal hosts perceive and respond to microbial cues.