The luminal environment of the epididymis participates in sperm maturation and impacts male fertility. It is dependent on the coordinated expression of many genes encoding proteins with a role in epithelial transport. We identified cis-regulatory elements for critical genes in epididymis function, by mapping open chromatin genome-wide in human epididymis epithelial (HEE) cells. Bioinformatic predictions of transcription factors binding to the regulatory elements suggested an important role for hepatocyte nuclear factor 1 (HNF1) in the transcriptional program of these cells. Chromatin immunoprecipitation and deep sequencing (ChIP-seq) revealed HNF1 target genes in HEE cells. In parallel, the contribution of HNF1 to the transcriptome of HEE cells was determined by RNA-seq, following siRNA-mediated depletion of both HNF1alpha and HNF1beta transcription factors. Repression of these factors caused differential expression of 1892 transcripts (902 were downregulated and 990 upregulated) in comparison to non-targeting siRNAs. Differentially expressed genes with HNF1 ChIP-seq peaks within 20 kb were subject to gene ontology process enrichment analysis. Among the most significant processes associated with down-regulated genes were epithelial transport of water, phosphate and bicarbonate, all critical processes in epididymis epithelial function. Measurements of intracellular pH (pHi) confirmed a role for HNF1 in regulating the epididymis luminal environment.