Abstract: Objective To investigate the radiosensitizing effect of Hedyotis diffusa(HDI) injection on esophageal cancer cells and related mechanism.Methods Esophageal cancer cells ECA-109 and TE-10 were treated with different concentrations of HDI for 24 h and 48 h, and the cell viability was detected by CCK-8 assay. The effect of HDI on the clone formation of ECA-109 and TE-10 cells was examined by the clone plate formation test. ECA-109 and TE-10 cells were divided into different groups, and their apoptosis and the expression of P38, ERK and JNK proteins were detected.Results HDI inhibited the survival rate of ECA-109 and TE-10 cells in a time and dose-dependent manner. The IC₅₀ of 24 h HDI was 66.56 ml/L and 56.52 ml/L for ECA-109 and TE cells, respectively. HDI inhibited the clone formation of ECA-109 and TE-10 cells in a dose-dependent manner. Compared with X-ray exposure, HDI+ ray treatment remarkably increased the apoptosis of ECA-109 and TE-10 cells in a dose-dependent manner(P<0.05). Compared with those treated with X-ray or HDI alone, ECA-109 and TE-10 cells showed significantly decreased levels of ERK, JNK and P38 proteins.Conclusions HDI can effectively improve the radiosensitivity of esophageal cancer cells, which may be related to reducing the phosphorylation levels of ERK, JNK and P38 in the MAPK pathway.