The objective of this study was to investigate the relationship between radiotherapy sensitivity, glutamine synthetase (GS), and oxidative stress (OS) in human hepatocellular carcinoma (HCC) cells. HCC cells were X-ray irradiated, and the effect of glutamine synthetase inhibition on the proliferative capacity of HCC cells was examined using the CCK-8 colony formation assay. Real-time quantitative PCR assays were used to detect the effect of L-methionine sulfoximine (MSO) on cellular glutamine synthetase expression levels and the efficiency of glutamine synthetase knockdown in HepG2 cells. Glutamine synthetase activity assay kit was used to detect the viability of glutamine synthetase in cells and tissues. Oxidative stress production was assayed using an oxidative stress assay kit. Subcutaneous xenografts were used to detect the effects of L-methionine sulfoximine and radiation on tumor growth in vivo. The results showed that the apparent cell proliferation capacity of HCC cells after glutamine synthetase inhibition was significantly reduced after radiotherapy, which was closely related to the increased production of oxidative stress after radiotherapy. Furthermore, the results of animal experiments also showed that the combination of L-methionine sulfoximine and radiation induced a stronger tumor suppressive effect and that L-methionine sulfoximine could act as a radiosensitizer after radiotherapy.