USP8 is a deubiquitinating enzyme that works as a regulator of endosomal sorting and vesicle morphology in cultured cells. Its function in vivo is, however, unknown as USP8 gene deletion leads to embryonic lethality. Previously we have shown that USP8 is highly expressed in male germ cells. These cells develop a peculiar acidic vesicle, indispensable for fertilization, the acrosome; in vivo USP8 might be involved in acrosomogenesis. The objective of this study was to test this hypothesis by determining if selective components of the early endosomal machinery interact functionally with USP8 during acrosomogenesis using protein-protein interaction assays and double/triple immunolabeling. Moreover, by exploiting the characteristic of USP8 that exhibits a microtubule interacting and transport (MIT) domain, we verified if USP8 effectively associates with spermatid microtubules by microtubule co-sedimentation and binding assays. USP8 was able to interact with spermatid ESCRT-0 complex and microtubule structures; USP8/ESCRT-0-labelled vesicles, monitored by fluorescence microscopy, were found to contribute to acrosome formation while USP8 directly linking, via the MIT domain, the labeled vesicles/developing acrosome to microtubules could favor both acrosome assembly and shaping. VPS54, the vacuolar sorting protein responsible for early endocytic retrograde transport, was here detected for the first time in male germ cells; VPS54 followed the intracellular route of USP8/ESCRT-0-labeled vesicles during acrosomogenesis. We concluded that in vivo USP8 has a role strongly associated with acrosome biogenesis and that the early endosome pathway is significantly involved in the process, which suggests that the acrosome could be a novel lysosome-related organelle