The ability of a spermatozoon to recognise and bind to the outer shell of an oocyte, the zona pellucida, is a key determinant of fertility potential. Fully differentiated spermatozoa only acquire the capacity for fertilisation after they have completed epididymal transit. This critical maturation process is achieved remarkably, in the complete absence of de novo protein translation. Instead, epididymal maturation is driven by the microenvironment sperm encounter within the lumen of the epididymal tubule. A key element of this dynamic environment are epididymosomes, small membrane-encapsulated vesicles that are secreted from the epididymal epithelial cells lining the lumen of the duct. These vesicles have been implicated in the direct transfer of diverse biomolecular cargo to the maturing gametes, including protein receptors putatively involved in downstream oocyte binding. In this study, we have performed comparative proteomic profiling of mouse epididymosomes isolated from different segments of the epididymis using mass tag based quantification via high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified, with 146 proteins being differentially accumulated between caput and corpus epididymosomes, and a further 344 differentially accumulated between corpus and cauda epididymosomes (i.e., fold change of ≤ -1.5 or ≥ 1.5; P < 0.05). A subset of the epididymosome proteins that have not been previously curated in current exosome databases, exhibit roles associated with the acquisition of sperm function including binding to the zona pellucida. Together with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data underpin the role of extracellular vesicles in coordinating post-testicular sperm maturation and ultimately the fertility potential of the spermatozoon. Understanding the roles of epididymosomes will advance our knowledge of male infertility and sperm dysfunction, while also opening potential avenues towards enhancing sperm quality for assisted reproductive technologies.