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Vitamin D (VD) is important for male reproduction in mammals and the VD receptor (VDR) and VD‐metabolizing enzymes are expressed in human spermatozoa. The VD‐inactivating enzyme CYP24A1 titrates the cellular responsiveness to VD, is transcriptionally regulated by VD, and has a distinct expression at the sperm annulus. Here, we investigated if CYP24A1 expression serves as a marker for VD metabolism in spermatozoa, and whether CYP24A1 expression was associated with semen quality. We included 130 men (53 healthy young volunteers and 77 subfertile men) for semen analysis and immunocytochemical (ICC) detection of CYP24A1. Another 40 men (22 young, 18 subfertile) were tested for in vitro effects of 1,25(OH)₂D₃ on intracellular calcium concentration ([Ca²⁺]_i) and sperm motility. Double ICC staining showed that CYP24A1 and VDR were either concomitantly expressed or absent in 80% of …