Radiotherapy amplifies p53 expression in cancer cells with wild-type (wt) p53. Blocking the negative regulators MDM2 and MDMX stabilizes p53 and may therefore potentiate radiotherapy outcomes. In this study, we investigate the efficacy of the novel anti-MDM2/X stapled peptide PM2 alone and in combination with external gamma radiation in vitro and in vivo PM2 therapy combined with radiotherapy elicited synergistic therapeutic effects compared with monotherapy in cells with wt p53 in both in vitro and in vivo assays, whereas these effects did not manifest in p53 -/- cells. Biodistribution and autoradiography of 125I-PM2 revealed high and retained uptake homogenously distributed throughout the tumor. In mice carrying wt p53 tumors, PM2 combined with radiotherapy significantly prolonged the median survival by 50%, whereas effects of PM2 therapy on mutant and p53 -/- tumors were negligible. PM2-dependent stabilization of p53 was confirmed with ex vivo immunohistochemistry. These data demonstrate the potential of the stapled peptide PM2 as a radiotherapy potentiator in vivo and suggest that clinical application of PM2 with radiotherapy in wt p53 cancers might improve tumor control.Significance: These findings contribute advances to cancer radiotherapy by using novel p53-reactivating stapled peptides as radiosensitizers in wild-type p53 cancers.