Spermatozoa released from the testes are functionally immature, lacking both motility and the potential to fertilise an ovum. These attributes are progressively acquired as they traverse the several meters of the epididymal tubule, a highly specialised region of the male reproductive tract. Since spermatozoa are both transcriptionally and translationally quiescent, this functional transformation is driven exclusively by the luminal microenvironment created by the combined secretory and absorptive of the lining epithelium. Central components of this microenvironment are epididymosomes, a heterogeneous population of small membrane bound vesicles that are released from the epididymal epithelium via an apocrine secretory mechanism. Similar to the extracellular vesicle population documented in other somatic tissues and bodily fluids, epididymosomes are beginning to emerge as attractive candidate vectors to facilitate the transfer of both proteomic and epigenetic information to spermatozoa. However, there remain fundamental challenges to this field of extracellular vesicle research. Not the least, is the development of robust and reproducible methods for epididymosome isolation and characterisation, particularly in the context of established laboratory models such as the rodents, where the scale of epididymal fluid recovery remains a particular challenge. Here, in an effort to address this limitation, we report the validation of a simple method of epididymosome isolation from differing segments of the mouse epididymis and the profiling of the both the proteomic and small non-protein-coding RNA (sRNA) content of these extracellular vesicles. Additionally, we provide the first direct evidence for the selective transfer of both protein and sRNA cargo between epididymosomes and mouse spermatozoa. Such data are of considerable interest in view of the potential role sRNA play in altering the sperm epigenome, and further, manipulation of this specific cargo may mediate direct consequences in offspring if the paternal linage encounters environmental insult(s).