Radiotherapy plays a more and more important role in the management of lung cancer. However, radioresistance still limits the long-term control of tumors. Exploration of the dynamic changes of metabolic reprogramming in surviving/resistant lung cancer cells exposed to increased doses of radiation is helpful to elucidate the metabolic mechanism of radiation resistance in lung cancer cells, to develop new targets for the clinical intervention and the early detection of radiation resistance. Lung cancer cells A549, H520 and H460 were treated in vitro with fractionated radiation (2 Gy) to a cumulative total dose of 40Gy, 60Gy or 80Gy. Cellular radiation sensitivity was verified by colony survival assay and comet experiments. Cell proliferation was determined by EdU assay. Transcriptome sequencing and metabolomic analysis were performed to identify differentially expressed genes and metabolites in radiation-resistant cells and their parental cells. Oil Red O staining was used to detect lipid droplet content. RT-PCR and WB were used to detect the expression of genes related to lipid metabolism. Lung cancer cell sublines that had been exposed to 20, 30 or 40 fractions of 2Gy had a significant increase in radioresistance as compared with their parental cells. Furthermore, this radioresistance of cells increased with the increase of previous radiation dose exposure. We found that lipid droplet deposition and the expression of lipid metabolism genes, such as fatty acid oxidation-related genes and glycerol metabolism-related genes, showed an increasing trend with previous radiation dose in surviving/resistant lung cancer cells exposed to radiation. Metabolomic analysis revealed that lipid metabolites such as glycerol and oxaloacetic acid also increased significantly. Inhibition of carnitine palmitoyltransferase 1A (CPT1A) by Etomoxir, a fatty acid oxidation inhibitor, significantly enhanced the radiosensitivity and decreased the DNA repair ability of various surviving/resistant lung cancer cells exposed to radiation. The surviving/resistant lung cancer cells generated by sequential irradiation provide models for future investigations of the dose-dependent mechanisms of radioresistance in a preclinical setting. Radiation dose-dependent lipid metabolism reprogramming may contribute to radiation survival/resistance in lung cancer cells. Support: 81572279, 2016J004, LC2016PY016, 2018CR033.