MDMX is a hetero dimeric partner of MDM2 and a critical regulator of p53. MDMX level is generally elevated in tumors with wild type p53 and contributes to p53 inactivation. MDMX degradation is controlled in part by MDM2-mediated ubiquitination. Here we show that MDMX turnover is highly responsive to changes in MDM2 level in non-transformed cells, but not in tumor cells. We found that loss of ARF expression, which occurs in most tumors with wild type p53, significantly reduces MDMX sensitivity to MDM2. Restoration of ARF expression in tumor cells enables MDM2 to degrade MDMX in a dose-dependent fashion. ARF binds to MDM2 and stimulates a second-site interaction between the central region of MDM2 and MDMX, thus increases MDMX-MDM2 binding and MDMX ubiquitination. These results reveal an important abnormality in the p53 regulatory pathway as a consequence of ARF deficiency. Loss of ARF during tumor development not only prevents p53 stabilization by proliferative stress, but also causes accumulation of MDMX that compromises p53 activity. This phenomenon may reduce the clinical efficacy of MDM2-specific inhibitors by preventing MDMX down regulation.