Purpose To assess the radio-sensitizing effect of the biguanide drug Metformin used alone or in combination with reactive oxygen species (ROS) modifying agents N-acetyl-L-cysteine (NAC) or emodin, and contrasted to the mitochondrial complex 1 inhibitor rotenone in altering the radiation responses of the p53 wild type SA-NH and p53 mutant FSa mouse tumor lines grown either in vitro or in vivo. Materials and Methods Tumor cells were grown to confluence in vitro and exposed to a single 4 Gy dose in the presence or absence of Metformin (5 mM) and ROS modifiers or to 10 Gy with or without Metformin as tumors in the flanks of C3H mice using a tumor growth delay assay. Results Both Metformin and rotenone protected SA-NH (P<0.001) while sensitizing FSa (P<0.001) to 4 Gy. Neither NAC nor emodin altered Metformin’s action. Metformin was also directly toxic to FSa cells (P=0.002). In contrast, in vivo Metformin (250 mg/kg) sensitized both SA-NH (9 day growth delay, P<0.05) and FSa (4 day growth delay, P<0.05) tumors if administered 1 h before irradiation. Conclusion Metformin effects on tumor cells measured under in vitro conditions may differ from those determined in vivo due to p53 and heterogeneous environmental factors.