Vitamin D has broad range of physiological functions and antitumor effects. 24-Hydroxylase, encoded by the CYP24A1 gene, is the key enzyme for degrading many forms of vitamin D including the most active form, 1,25D₃. Inhibition of CYP24A1 enhances 1,25D₃ antitumor activity. To isolate regulators of CYP24A1 expression in prostate cancer cells, we established a stable prostate cancer cell line PC3 with CYP24A1 promoter driving luciferase expression to screen a small molecular library for compounds that inhibit CYP24A1 promoter activity. From this screening, we identified, 4,5,6,7-tetrabromobenzimidazole (TBBz), a protein kinase CK2 selective inhibitor as a disruptor of CYP24A1 promoter activity. We show that TBBz inhibits CYP24A1 promoter activity induced by 1,25D₃ in prostate cancer cells. In addition, TBBz downregulates endogenous CYP24A1 mRNA level in TBBz-treated PC3 cells. Furthermore, siRNA-mediated CK2 knockdown reduces 1,25D₃-induced CYP24A1 mRNA expression in PC3 cells. These results suggest that CK2 contributes to 1,25D₃-mediated target gene expression. Finally, inhibition of CK2 by TBBz or CK2 siRNA significantly enhances 1,25D₃-mediated antiproliferative effect in vitro and in vivo in a xenograft model. In summary, our findings reveal that protein kinase CK2 is involved in the regulation of CYP24A1 expression by 1,25D₃ and CK2 inhibitor enhances 1,25D₃-mediated antitumor effect. Cancer Res; 73(7); 2289–97. ©2013 AACR.