STUDY HYPOTHESIS: Region-specific transcriptional profiling of tissues and cultured epithelial cells from the human epididymis will predict functional specialization along the duct. STUDY FINDING: We identified the molecular signature driving functions of the caput, corpus and cauda epithelium, and determined how these differ to establish the regional differentiation of the organ. WHAT IS KNOWN ALREADY: The epithelium lining the human male genital ducts has a critical role in fertility. In particular, it controls the luminal environment in the epididymis, which is required for normal sperm maturation and reproductive competence. Studies in many animal species have largely informed our understanding of the molecular basis of epididymis function. However, there are substantial differences between species. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Using RNA sequencing on biological replicates, we described gene expression profiles for tissue from each region of the epididymis and cultured epithelial cells derived from these regions. Bioinformatic tools were then utilized to identify differentially expressed genes (DEGs) between tissues and cells from the caput, corpus and cauda. MAIN RESULTS AND THE ROLE OF CHANCE: The data showed that the caput is functionally divergent from the corpus and cauda, which have very similar transcriptomes. Interrogation of DEGs using gene ontology process enrichment analyses showed that processes of ion transport, response to hormone stimulus and urogenital tract development are more evident in the caput, while defense response processes are more important in the corpus/cauda. Consistent with these regional differences in epididymis function, we observed differential expression of transcription factors in the caput and corpus/cauda. LIMITATIONS, REASONS FOR CAUTION: Cultured caput, corpus and cauda cells may not faithfully represent the same cells in the intact organ, due to loss of hormonal signals from the testis and communication from other cell types. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide a molecular characterization that will facilitate advances in understanding human epididymis epithelium biology in health and disease. They may also reveal the mechanisms coordinating epididymis luminal environment and sperm maturation. LARGE SCALE DATA: Data deposited at http://www.ncbi.nlm.nih.gov/geo/GSE72986. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by the National Institutes of Health: R01HD068901 (PI: A.H.). The authors declare no conflict of interest.