The mammalian epididymis is a highly specialized region of the male reproductive tract that is lined with a continuous layer of epithelial cells that display a remarkable level of regionalized secretory and absorptive activity. The luminal environment created by this combined secretory and absorptive activity is directly responsible for promoting the functional maturation of spermatozoa and their maintenance in a quiescent and viable state prior to ejaculation. This study was designed to identify the complement of microRNAs (miRNAs) that are expressed within the mouse epididymal epithelial cells and the maturing populations of spermatozoa. Through the use of Next Generation Sequencing technology we have demonstrated that both epididymal epithelial cells and spermatozoa harbour a complex repertoire of miRNAs that have substantially different expression profiles along the length of the tract. These data, deposited in the Gene Expression Omnibus (GEO) with the accession numbers GSE70197 and GSE70198, afford valuable insight into the post-transcriptional control of gene expression within the epididymis and provide the first evidence for the dynamic transformation of the miRNA content of maturing sperm cells. Ultimately such information promises to inform our understanding of the aetiology of male infertility. Herein we provide a detailed description of the methodology used to generate these important data.