Mammalian spermatozoa acquire functionality during epididymal maturation, and the ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to assess the effects of epididymal maturation, ejaculation and in vitro capacitation on sperm viability, acrosome integrity, mitochondrial activity, membrane fluidity, and calcium influx, both as indicators of capacitation status and sperm motility. Results indicated that boar spermatozoa acquired the ability to move in the epididymal corpus; however, their motility was not linear until the ejaculation. Epididymal spermatozoa showed low membrane fluidity and intracellular calcium content; ejaculation led to an increased calcium content, while membrane fluidity showed no changes. Acrosome integrity remained constant throughout the epididymal duct and after ejaculation and in vitro capacitation. The frequency of viable spermatozoa with intact mitochondrial sheath was higher in caput and ejaculated samples than in corpus and cauda samples, whereas the frequency of spermatozoa with high membrane potential was significantly lower in cauda samples. In vitro capacitation resulted in a decreased frequency of viable spermatozoa with intact mitochondrial sheath and an increased frequency of spermatozoa with high membrane potential in ejaculated samples. These results indicated that both epididymal maturation and ejaculation are key events for further capacitation, because only ejaculated spermatozoa are capable of undergoing the set of changes leading to capacitation.