PURPOSE: We evaluated the usefulness of serum 25-hydroxyvitamin D as a marker of aggressive prostate cancer and for active surveillance compared to PHI (Prostate Health Index). MATERIALS AND METHODS: Of 480 prospectively biopsied men 222 had prostate cancer and 258 had no evidence of malignancy. In all men prostate specific antigen was less than 20 ng/ml. We measured 25-hydroxyvitamin D, prostate specific antigen, free prostate specific antigen and -2proPSA using a commercially available immunoassay system. PHI was calculated according to the equation, -2proPSA/free prostate specific antigen × √PSA. We determined 25-hydroxyvitamin D using a 2-step competitive binding immunoenzymatic vitamin D assay. RESULTS: The 25-hydroxyvitamin D concentrations were not associated with Gleason grade according to the 2014 ISUP (International Society of Urological Pathology) consensus conference Gleason grading system. PHI values were higher with increasing Gleason grade. Median 25-hydroxyvitamin D did not differ between men with prostate cancer vs no evidence of malignancy (50.6 vs 48.2 nmol/l, p = 0.192) or in ISUP Gleason subgroups despite seasonal variations of 25-hydroxyvitamin D. However, PHI values significantly differed between the subgroup with no evidence of malignancy and all Gleason subgroups (p <0.0001). The ROCs of all men revealed an advantage of PHI over 25-hydroxyvitamin D (AUC 0.78 vs 0.535, p <0.0001). PHI could also significantly better separate patients with no evidence of malignancy from those with nonaggressive disease (ISUP Gleason grade 1) from those with aggressive prostate cancer (ISUP Gleason grades 2-5). CONCLUSIONS: It remains highly improbable that 25-hydroxyvitamin D could be used as decision or selection marker for aggressive prostate cancer or for active surveillance compared to accepted markers, as recently suggested.