Sperm undergo maturation acquiring progressive motility and the ability to fertilize oocytes through exposure to the components of the epididymal fluid (EF). Although the establishment of a calcium (Ca2+) gradient along the epididymis has been described, its direct effects on epididymal function remain poorly explored. Regucalcin (RGN) is a Ca2+-binding protein, regulating the activity of Ca2+-channels and Ca2+-ATPase, for which a role in male reproductive function has been suggested. This study aimed at comparing the morphology, assessed by histological analysis, and function of epididymis, by analysis of sperm parameters, antioxidant potential and Ca2+ fluxes, between transgenic rats overexpressing RGN (Tg-RGN) and their wild-type littermates. Tg-RGN animals displayed an altered morphology of epididymis and lower sperm counts and motility. Tissue incubation with 45Ca2+ showed also that epididymis of Tg-RGN displayed a diminished rate of Ca2+-influx, indicating unbalanced Ca2+ concentrations in the epididymal lumen. Sperm viability and the frequency of normal sperm, determined by the one-step eosin-nigrosin staining technique and the Diff-Quik staining method, respectively, were higher in Tg-RGN. Moreover, sperm of Tg-RGN rats showed a diminished incidence of tail defects. Western blot analysis demonstrated the presence of RGN in EF as well as its higher expression in the corpus region. The results presented herein demonstrated the importance of maintaining Ca2+-levels in the epididymal lumen and suggest a role for RGN in sperm maturation. Overall, a new insight into the molecular mechanisms driving epididymal sperm maturation was obtained, which could be relevant to development of better approaches in male infertility treatment and contraception.