Diminished expression of NKX3.1 is associated with prostate cancer progression in humans, and in mice, loss of nkx3.1 leads to epithelial cell proliferation and altered gene expression patterns. The NKX3.1 amino acid sequence includes multiple potential phosphoacceptor sites for protein kinase CK2. To investigate posttranslational regulation of NKX3.1, phosphorylation of NKX3.1 by CK2 was studied. In vitro kinase assays followed by mass spectrometric analyses demonstrated that CK2 phosphorylated recombinant NKX3.1 on Thr89 and Thr93. Blocking CK2 activity in LNCaP cells with apigenin or 5,6-dichlorobenzimidazole riboside led to a rapid decrease in NKX3.1 accumulation that was rescued by proteasome inhibition. Replacing Thr89 and Thr93 with alanines decreased NKX3.1 stability in vivo. Small interfering RNA knockdown of CK2α′ but not CK2α also led to a decrease in NKX3.1 steady-state level. In-gel kinase assays and Western blot analyses using fractionated extracts of LNCaP cells demonstrated that free CK2α′ could phosphorylate recombinant human and mouse NKX3.1, whereas CK2α′ liberated from the holoenzyme could not. These data establish CK2 as a regulator of NKX3.1 in prostate tumor cells and provide evidence for functionally distinct pools of CK2α′ in LNCaP cells.