Newly formed sperm exiting the testis cannot fertilise an egg as they need to acquire motility and fertilising proteins via transit through the epididymis. Since sperm lack the ability to translate their own proteins, they rely on external sources to provide these proteins. Epithelial cells of the epididymis release extracellular vesicles (EVs, also known as “epididymosomes”) into the lumen which contain these essential proteins and are taken up by the sperm. We have previously shown that Arrdc4 is important for the biogenesis and protein targeting of plasma membrane derived EVs. Arrdc4-/- male mice were observed to be less efficient breeders, producing either no or less than average number of pups per breeding when paired with heterozygous females, compared with heterozygous breeding pairs or with Arrdc4-/- female mice paired with heterozygous males. We then analysed the sperm from Arrdc4-/- mice and have found that sperm develop normally through the testis, with stained testis sections showing no difference in sperm development and no difference in daily sperm production or cauda sperm concentration. Using Computer Assisted Sperm Analysis, we found that cauda sperm have a reduced motility, and also reduced ability to fertilise oocytes as measured using in vitro fertilisation when compared to wild type sperm. Using mass spectrometry, caput and cauda sperm proteomes were compared between wild type and Arrdc4-/- mice. We found that Arrdc4-/- cauda sperm have a reduction in many proteins that are gained through epididymal transit by wild type sperm, specifically proteins involved in motility, energy production and the acrosome reaction (as identified using FunRich pathway analysis), and these are currently being validated. We have also found that Arrdc4-/- epididymal epithelial cells have a reduction in both EV production and EV size, as measured by Nanoparticle Tracking Analysis and transmission electron microscopy, suggesting a loss of the larger, plasma membrane derived vesicles. We hypothesise that Arrdc4 may be important for regulating EV production in the epididymis which is required for proper sperm maturation.