Spermatozoa acquire functional competence as they descend through the epididymis, a highly specialised region of the male reproductive tract. A characteristic feature of the epididymis is its ability to create highly regionalised microenvironments that promote sperm maturation. Although the molecular mechanisms through which these dynamic microenvironments are generated remains to be fully elucidated, recent studies have implicated the RNA interference pathway as a key regulator of epididymal gene expression profiles. Hence, in this study we have applied next generation sequencing technology to explore the microRNA (miRNA) signature of mouse epididymal epithelial cells and that of spermatozoa maturing within the lumen of the duct. In doing so we have demonstrated substantial segmental differences in the expression of epithelial miRNAs, as well as providing the first evidence for the post-testicular modification of miRNA profiles in spermatozoa under normal physiological conditions. In total, 370 miRNAs were identified in whole epididymal tissue, 218 of which proved to be unique to epithelial cells, including a substantial portion that putatively regulate canonical signalling pathways involved in maintenance of cellular development, proliferation and cell death. Similarly, an impressive profile of some 295 miRNAs were identified in mouse spermatozoa as they enter the epididymis. This profile was however, dynamically altered following the apparent loss of 113 and acquisition of a further 115 miRNAs between the proximal (caput) and terminal (cauda) regions of the duct. Bioinformatic analyses revealed that the miRNAs present in mature spermatozoa mapped predominantly to NF-κB and TGFβ signalling pathways that have been implicated in conditioning of the peri-conceptual environment within the female reproductive tract and in regulating embryonic development. Collectively, the data presented herein provide the first evidence in support of a key role for miRNA regulation of epididymal sperm maturation in the mouse model.