In this study, using radiation-tolerant mouse melanoma cell line B16 as research model , cells were first radiated with sublethal doses of X-ray radiation; then cellular metabolites were collected, which were further analyzed by 600 MHz 1H NMR to screen differentially expressed metabolites. Following bioinformatic analysis for those quantified metabolites, potential targets of disturbed metabolic pathway can be used for radiosensitization study to discover high efficacy drugs for sensitization in radiotherapy. The underlying mechanism of metabolome changes and radiation resistance in B16 cells was discussed.