Male fertility is routinely evaluated by semen analysis, although semen quality variables such as; sperm count, motility and morphology have low predictive value for spontaneous pregnancies and fertility treatment outcomes. Vitamin D has been suggested to be beneficial for male reproduction. The vitamin D receptor and the vitamin D inactivating enzyme CYP24A1 are co-expressed in high quality sperm. Presence of CYP24A1 at the annulus of human sperm can distinguish between sperm from healthy and infertile men with high specificity and is positively correlated with semen quality. The high expression level in the testis of FAM57B2, which is activated by 24,25OH2D3, indicates an uncharacterized biological role for CYP24A1 in male reproduction. Moreover, activated vitamin D has been shown to induce sperm motility and promote fertilization in vitro. Here, we prospectively investigated whether the fraction of CYP24A1 positive sperm was a better predictor of clinical pregnancy than semen analysis by including 240 fertility treatments (169 couples) from a single fertility centre in Denmark. ROC-curve based analysis showed that the percentage of sperm expressing CYP24A1 was a better predictor of successful pregnancy outcome after intrauterine inseminations (IUI) than both sperm concentration and motility (p < 0.05). Interestingly, samples with CYP24A1 staining >67% of the sperm increased the likelihood of achieving pregnancy 4-fold after IUI compared with samples having fewer sperm with detectable CYP24A1 (p < 0.05). Neither CYP24A1 nor any of the other assessed semen quality variables were predictive for the treatment outcome of the more invasive assisted reproductive techniques (IVF and ICSI). In conclusion, our results provide proof of principle for a CYP24A1-based sperm test to improve fertility outcome for infertile patients referred for IUI and supports a role for vitamin D metabolites during fertilization.