Homeodomain-interacting protein kinase-2 (HIPK2) is a serine/threonine kinase involved in transcriptional regulation and apoptosis. The transcriptional corepressor CtBP (carboxyl-terminal binding protein) also plays a fundamental role in these processes. Our previous studies indicate that HIPK2 participates in a pathway of UV-triggered CtBP clearance that results in cell death. HIPK2 phosphorylates CtBP at Ser-422 in vitro. We developed a Ser-422 phospho-specific antibody to demonstrate that CtBP is phosphorylated on this residue in response to UV irradiation. HIPK2 knock-down blocked the UV-induced Ser-422 phosphorylation and degradation. The proteasomal inhibitor MG-132 treatment increased levels of ubiquitinated CtBP, which was induced by UV. Interference with HIPK2 function via the kinase-dead mutant decreased CtBP ubiquitination. Furthermore, a phosphopeptide spanning Ser-422 blocked UV-triggered CtBP degradation, confirming that Ser-422 phosphorylation marks CtBP for clearance. Consequently, interference with HIPK2 action in H1299 cells rescued UV-triggered apoptosis.