Spermatozoa released from the testis are functionally incompetent and incapable of fertilising an oocyte. These functional attributes are acquired progressively as the sperm are conveyed through the epididymis and appear to be driven by extrinsic factors present within the complex intraluminal milieu. This dynamic microenvironment is, in turn, created by the combined secretory and absorptive activity of the epididymal epithelium and displays a remarkable level of regionalisation. While the factors responsible for the regulation of this environment are yet to be fully resolved, recent evidence has highlighted a novel and potentially important role for microRNAs (miRNAs). miRNAs are small non-coding RNAs that interact with several proteins to form an active RNA-induced silencing complex (RISC). The presence of miRNAs enables RISC to bind to specific target mRNAs and thereby regulate post-transcriptional gene expression. To begin to investigate the role of miRNAs in promoting sperm maturation we have focused on determining the expression profile of miRNAs and their associated RISC proteins, DICER1 and ARGONAUTE2 (AGO2), in the mouse epididymis. For this purpose we have used a combination of RNAseq, qPCR, and immunolocalisation studies. Our collective data indicates that several miRNAs, including miR223 and mIR15a-3p, are differentially expressed in both the epithelium and in spermatozoa sampled from the different regions of the epididymis. Similarly, we have shown that Dicer 1 mRNA expression is significantly increased in both the epithelial cells and spermatozoa from the distal regions of the epididymis. Interestingly, DICER1 and AGO2 proteins were also detected in spermatozoa and shown to be dynamically repositioned as the cells migrated through the epididymis, culminating in strong co-localisation in the head of mature caudal spermatozoa. Taken together, these data support an important role for miRNA in the regulation of epididymal function and raise the intriguing prospect that a subset of miRNAs may be transferred to the maturing spermatozoa.