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Objective: To investigate the killing effect of low-temperature plasma (LTP) on HepG2, A549 and HeLa cell lines and explore its possible mechanism. Methods: The inhibitory effect of LTP on the proliferation of HepG2, A549 and HeLa cells was determined by MTT assay. Transmission electron microscopy was used to observe the ultrastructural changes of HepG2, A549 and HeLa cells treated with LTP. Cell apoptosis was detected by Muse cytometry. Western blot was used to detect the expression of apoptosis-related proteins. Results: The survival rates of LTP-irradiated HepG2 cells (irradiated for 107 s), HeLa cells (irradiated for 121 s) and A549 cells (irradiated for 127 s) were 50%. LTP destroyed the ultrastructure of HepG2, A549 and HeLa cells to different degrees, showing nuclear fragmentation and organelle damages. The apoptosis rates of the three cell lines were increased at 24 h after exposure to LTP for 1/6 IC 50 irradiation time. Furthermore, LTP irradiation also suppressed the protein expression of Bcl-2 and XRCC1 and increased that of Bax. Conclusions: LTP has an obvious killing effect on HepG2, A549 and HeLa cancer cell lines. This effect may be related to the induction of cell apoptosis and inhibition of DNA repair.