IGFBP-3 promotes apoptosis by both IGF-dependent and -independent mechanisms. We have previously reported that phosphorylation of IGFBP-3 (S156) by DNA-PK enhances its nuclear accumulation, and is essential for its ability to interact with RXR and induce apoptosis in cultured prostate cancer cells. Using specific chemical inhibitors and siRNA, we demonstrate that preventing CK2 activation enhanced the apoptotic potential of IGFBP-3. We mapped potential CK2 phosphorylation sites in IGFBP-3 to S167 and S175, and identified that wtIGFBP-3 and IGFBP-3-S175A induced apoptosis to a comparable extent. In contrast, IGFBP-3-S167A was far more potently apoptosis-inducing due to inability to undergo CK2-phosphorylation. Pre-treatment of 22RV1 cells with IGFBP-3-siRNA also limits the ability of high doses of CK2 inhibitor to induce apoptosis. These effects can be reversed by the addition of exogenous IGFBP-3 protein suggesting reciprocal regulation of cell survival and apoptosis by IGFBP-3 and CK2. These studies reveal multi-site phosphorylation of IGFBP-3 that both positively and negatively regulate its apoptotic potential. Understanding such intrinsic regulation of IGFBP-3 action may enhance the development of potential cancer therapies.