MDMX is a homolog of MDM2 that is critical for regulating p53 function during mouse development. MDMX degradation is regulated by MDM2-mediated ubiquitination. Whether there are other mechanisms of MDMX regulation is largely unknown. We found that MDMX binds to the casein kinase 1 alpha isoform (CK1α) and is phosphorylated by CK1α. Expression of CK1α stimulates the ability of MDMX to bind to p53 and inhibit p53 transcriptional function. Regulation of MDMX-p53 interaction requires CK1α binding to the central region of MDMX and phosphorylation of MDMX on serine 289. Inhibition of CK1α expression by isoform-specific small interfering RNA (siRNA) activates p53 and further enhances p53 activity after ionizing irradiation. CK1α siRNA also cooperates with DNA damage to induce apoptosis. These results suggest that CK1α is a functionally relevant MDMX-binding protein and plays an important role in regulating p53 activity in the absence or presence of stress.