There is an increasing interest in determining the role of ribosomal proteins (RPs) in the regulation of MDM2-p53 pathway in coordinating cellular response to stress. Herein, we report a novel regulatory role of ribosomal protein S25 (RPS25) in MDM2-mediated p53 degradation and a feedback regulation of S25 by p53. We demonstrated that S25 interacted with MDM2 and inhibited its E3 ligase activity, resulting in the reduction of MDM2-mediated p53 ubiquitination and the stabilization and activation of p53. S25, MDM2 and p53 formed a ternary complex following ribosomal stress. The nucleolar localization and MDM2-binding domains of S25 were critical for its role in MDM2-mediated p53 regulation. Knockdown of S25 by siRNA attenuated the induction and activation of p53 following ribosomal stress. S25 stabilized and cooperated with MDMX to regulate MDM2 E3 ligase activity. Furthermore, S25 was identified to be a transcriptional target of p53; p53 directly bound to S25 promoter and suppressed S25 expression. Our results suggest that there is a S25-MDM2-p53 regulatory feedback loop, which may have an important role in cancer development and progression.