Although the recently identified Pirh2 protein is known as a p53-induced ubiquitin-protein E3 ligase, which negatively regulates p53, the detailed mechanism underlying the regulation of Pirh2 remains largely unknown. Here, we demonstrate that while Pirh2 is mostly detected in the phosphorylated form in normal tissues, it is predominantly present in the unphosphorylated form in majority of tumor cell lines and tissues examined. Phosphorylated Pirh2 is far more unstable than its unphosphorylated form. We further identified that Calmodulin-dependent kinase II (CaMK II) phosphorylates Pirh2 on residues Thr-154 and Ser-155. Phosphorylation of Pirh2 appears to be regulated through cell cycle-dependent mechanism. CaMK II-mediated Pirh2 phosphorylation abrogates its E3 ligase activity toward p53. Together, our data suggest that phosphorylation of Pirh2 may act as a fine-tuning to maintain the balance of p53-Pirh2 autoregulatory feedback loop, which facilitates the tight regulation of p53 stability and tumor suppression.