Epididymosomes are small membrane vesicles that are secreted by epididymal epithelial cells and are involved in posttesticular sperm maturation. Although their role in protein transfer to the sperm membrane is well documented, we report their capacity to transport microRNAs (miRNAs), which are potent regulators of posttranscriptional gene expression. Using a microperfusion technique combined with a global microarray approach, we demonstrated that epididymosomes from two discrete bovine epididymal regions (caput and cauda) possess distinct miRNA signatures. In addition, we also established that miRNA repertoires contained within epididymosomes differ from those of their parent epithelial cells, suggesting that miRNA populations released from the cells may be selectively sorted. Binding of DilC12-labeled epididymosomes to primary cultured epididymal cells was measured by flow cytometry, and the results indicated that epididymosomes from the median caput and their miRNA content may be incorporated into distal caput epithelial cells. Overall, these findings reveal that distinct miRNA repertoires are released into the intraluminal fluid in a region-specific manner and could be involved in a novel mechanism of intercellular communication throughout the epididymis via epididymosomes.