Radiation therapy can generate free radicals and reactive oxygen species (ROS) from X-ray radiation, which induces DNA damage in M-phase and G2-phase cells and subsequently blocks cells in the G2/M phase. By inducing cell cycle arrest–mediated apoptosis, cancer cells are killed with radiation therapy [31–33]. Nevertheless, it has been shown that multiple mechanisms lead to cancer tolerance of radiation therapy (e.g., anti-apoptosis , DNA damage repair , cell cycle regulation ). Previous studies indicated that radiation had little impact on the B16 cell cycle, and the observation of γH2AX foci showed that the ratio of DNA double strand break in radiated B16 cells was higher than that of untreated cells . Impaired DNA double-strand break repair by chemical castration in prostate cancer had an improved response to radiotherapy . In this study, the content of glycine in radiated B16 cells was increased. It is known that glycine can react with 5,10-methylenetetrahydrofolate by catalysis of glycine hydroxymethyltransferase to generate serine and tetrahydrofolic acid, and the latter is an important coenzyme of DNA synthesis process. This conclusion suggested that radiation tolerance in B16 cells may arise from DNA damage repair. Accordingly, by activating specific metabolic pathways to block DNA damage repair, radiation-tolerant cancer cells can be killed, which may possibly be an effective way of radiotherapy sensitization.