Mammalian spermatogenesis involves a series of events during which germ cells differentiate into immature sperm cells. Following spermatogenesis in the seminiferous tubules, sperm undergo post-testicular maturation where they suffer extensive remodelling of their proteome culminating in the acquisition of forward motility and fertilizing ability. Despite the advances, the proteomic landscape of mammalian testicular and epididymal sperm cells remains largely uncharacterized. Shotgun proteomics was used to compare proteomes of bull testicular, caput and cauda epididymal spermatozoa. This is crucial for understanding the physiological mechanisms that lead to sperm competence.