To explore the therapeutic effect of metformin combined with single high-dose radiotherapy in mice with non-small cell lung cancer and the mechanism involved. Invitro cell test: the cytotoxicity of metformin under different oxygen and glucose contents was evaluated using cell survival curves. Invivo xenograft tumor test: NSCLC-A549 cells were injected into mice, which were then divided into the following five groups: control group, metformin group, single high-dose radiotherapy group, metformin 1 h before radiotherapy group, and metformin 24 h after radiotherapy group. Growth of the tumor was evaluated in each group, and the blood flow and histology of the tumor tissues were analyzed. According to the invitro test results, metformin was the most cytotoxic under hypoxic and low-glucose conditions. The in vivo results showed that the tumor volume was the smallest in the mouse groups administered metformin 1 h before high-dose radiotherapy and 24 h after radiotherapy ((0.47±0.12) cm³ and (0.49±0.21) cm³, respectively). Blood flow analysis showed that single high-dose radiotherapy destroyed the micro-vessels of tumor tissue and reduced the perfusion of oxygen and glucose. Histological analysis revealed that cells in hypoxic and hypoglycemic area of the tumor tissue center were preferentially killed by metformin, whereas there were viable cells in the surrounding well perfused areas. Metformin combined with single high-dose radiotherapy enhanced the toxicity by changing tumor cell metabolism. These results provide theoretical and data support for the future use of metformin combined with stereotactic body radiotherapy.