The spectrum for vitamin D (VD) mediated effects has expanded in recent years. Activated VD (1,25(OH)(2)D(3)) binds to the VD receptor (VDR) and mediates non-genomic effects through the alternative ligand binding-pocket (VDR-ap) or regulates gene transcription through the genomic binding-pocket. VDR and VD-metabolizing enzymes are expressed in human testis, male reproductive tract and mature spermatozoa, and VD is considered important for male reproduction. Expression of the VD-inactivating enzyme CYP24A1 at the annulus of human spermatozoa distinguish normal and infertile men with high specificity, and CYP24A1 expression is positively correlated with all semen variables and suggested as a marker for both semen quality and VD responsiveness. Moreover, spermatozoa are transcriptionally silent and are therefore a unique model to study non-genomic effects. 1,25(OH)(2)D(3) induced a rapid increase in intracellular calcium concentration [Ca(2+)](i) in human spermatozoa. The [Ca(2+)](i) increase was abrogated by the non-genomic VDR antagonist 1ß,25(OH)(2)D(3), while the specific agonist for VDR-ap (JN) increased [Ca(2+)](i) with similar kinetics as 1,25(OH)(2)D(3). The rise in [Ca(2+)](i) originated as a Ca(2+)-release from intracellular stores since inhibition of phospholipase-C diminished the 1,25(OH)(2)D(3) mediated Ca(2+) response, while suspending spermatozoa in a nominally Ca(2+)-free medium did not affect the VD mediated Ca(2+) rise. The spatio-temporal kinetics of the VD-response differed from the progesterone-mediated increase in [Ca(2+)](i) as the VD-mediated Ca(2+) rise was not observed in the tail region and was independent of extracellular Ca(2+). A functional role of the VD-mediated Ca(2+) increase was supported by showing that 1,25(OH)(2)D(3) increased sperm motility and induced the acrosome reaction in vitro.