African American (AA) men are disproportionately affected by both prostate cancer (PCa) and vitamin D deficiency compared with European American (EA) men. Vitamin D deficiency is linked to increased PCa aggressiveness and mortality. Therefore, it has been hypothesized that vitamin D deficiency may contribute to the PCa disparity between AA and EA men. We studied a cross sectional group of 60 PCa patients (AA, = 31; EA, = 29) who underwent radical prostatectomy. Vitamin D metabolites 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)D) were measured in the serum and tissue by uHPLC-MS-MS. Tissue was laser capture microdissected, and gene expression was quantified by microarray. DNA isolated from whole blood was genotyped for West African ancestry markers and vitamin D-related SNPs. Serum concentrations of 25(OH)D were lower in AAs, but concentrations of 1,25(OH)D in the prostate tissue were higher compared with EAs. Expression of the vitamin D receptor was higher in prostate tissue from AAs. Expression of the extracellular receptor of vitamin D binding protein, , was positively associated with West African ancestry and inversely associated with tissue 25(OH)D concentrations in AAs. The relationships between vitamin D binding protein and vitamin D metabolites suggest that the prohormone is actively transported into the prostate, followed by intraprostatic conversion to the active hormone, rather than passive diffusion. These findings support the presence of a compensatory response in prostate tissue to vitamin D deficiency in AAs and reveal a previously unknown complexity involving tissue distribution of vitamin D metabolites. Department of Defense Prostate Cancer Research Program Idea Award for Disparities Research PC121923 (LN and RK) and the NIH 1R01MD007105 (RK).