The synthetic peptide TAT-RasGAP317-326 has been shown to potentiate the efficacy of anti-cancer drugs. In this study, we explored the action of TAT-RasGAP317-326 when combined with radiation by investigating its radiosensitizing activity in vitro and in vivo. To investigate the modulation of intrinsic radiosensitivity induced by TAT-RasGAP317-326, clonogenic assays were performed using four human cancer cell lines, HCT116 p53(+/+) (ATCC: CCL-247), HCT116 p53(-/-), PANC-1 (ATCC: CRL-1469) and HeLa (ATCC: CCL-2), as well as one nontumor cell line, HaCaT (CLS: 300493). Next, to investigate tumor growth delay after irradiation, HCT116 cell lines were selected and xenografted onto nude mice that were then treated with TAT-RasGAP317-326 alone or in combination with radiation or cisplatin. Afterwards, cell cycle and death modulation were investigated by quantification of micronuclei and apoptosis-related protein array. TAT-RasGAP317-326 radiosensitized all four human carcinoma cell lines tested but displayed no effect on normal cells. It also displayed no effect when administered as monotherapy. This radiosensitizing effect was confirmed in vivo in both p53-positive and p53-negative HCT116 xenografts. TAT-RasGAP317-326 combined with radiation enhanced the number of cells in S phase and subsequently delayed cell death, but had almost no effect on major apoptosis-related proteins. TAT-RasGAP317-326 is a radiosensitizing agent that acts on carcinoma cells and its radiosensitizing effect might be mediated, at least in part, by the enhancement of mitotic cell death.