Abstract: Objective：To investigate the synergistic effect of raltitrexed combined with radiotherapy on Hep-2 cells and its potential mechanism.Methods：CCK-8 assay was used to determine the cell viability after Hep-2 cells were treated with different concentrations of raltitrexed. Hep-2 cells were randomly divided into the control group, the raltitrexed group, the radiation group, and raltitrexed + radiation group. Cell survival curves were fitted with a single-hit multi-target model, cell viability and sensitization enhancement ratio(SNR) of the 4 groups were detected, and apoptosis and cycle changes of Hep-2 cells in 4 groups were detected by flow cytometry.Results：CCK-8 assay proved that raltitrexed could suppress cell viability in a dose-dependent manner. The single-hit multi-target model showed that the SER of mean death dose (D0) was 1.272.Furthermore, the apoptosis rate and the number of Hep-2 cells at G2/M phase were significantly increased in the group treated with the combination of raltitrexed and X-ray as compared to the group treated with X-ray alone (P<0.05).Conclusions：The inhibitory effect of raltitrexed combined with X-ray on human laryngeal cancer Hep-2 cells may be related to the induction of tumor cell apoptosis and the increase of G2/M phase arrest.