Objective To determine the role of the DNA damage response (DDR) pathway in calcification of human vascular smooth muscle cell (HVSMC) induced by high calcium and phosphorus.Methods HVSMCs were divided into the control group, model group, ataxia-telangiectasia mutated inhibitor (iATM) group, and poly (ADP-ribose) polymerase inhibitor (iPARP) group, and were cultured for 12 days. Qualitative and quantitative analyses of HVSMC calcification in the four groups were performed via Alizarin red S staining and o-Cresolphthalein chromogenic method, respectively. The DNA damage was measured via the comet assay. The phosphorylation level of histone γH2AX was detected by Western blotting and immunofluorescence, while the level of 8-hydroxy-2'-deoxyguanosine (8-OHDG) was detected by enzyme-linked immunosorbent assay (ELISA). Besides, the cell viability of the four groups was analyzed by Nucleo Counter ? NC-3000 ? Advanced Cell Analyzer.Results From the 9th day on, intracellular calcium deposition as shown under the light microscope and by the Alizarin red S staining occurred in the model group in comparison to the control group, and the calcium deposition was obvious on the 12th day. The ratio of the level of calcium to that of proteins on the 3rd, 6th, 9th, and 12th day was compared between the control group and the model group, which suggested that the ratio of the level of calcium to that of proteins was different among the time points (F = 168.970, P = 0.000) and between the groups (F = 203.040, P = 0.000), where the ratio of the level of calcium to that of proteins and the degree of calcification were greater in the model group. In addition, the change trends of the ratio of the level of calcium to that of proteins was different between the two groups (F =13.213, P = 0.000). On the 12th day of culture, the degree of calcification as shown by the Alizarin red S staining was higher in the model group than in the control group, whereas the degree of calcification in the iATM group and the iPARP group was lower than that in the model group. The calcium assay via the o-Cresolphthalein chromogenic method demonstrated that the ratio of the level of calcium to that of proteins was lower in the iATM group and the iPARP group than in the model group (P < 0.05). The comet assay found that there were greater numbers of cells bearing DNA damage in the model group than in the control group since the 9th day. The percentage of cells bearing DNA damage on the 3rd, 6th, 9th, and 12th day of culture was compared between the control group and the model group, and the results exhibited that the percentage of cells bearing DNA damage was different among the time points (F = 13.141, P = 0.000) and between the groups (F = 121.521, P = 0.000). Specifically, the percentage of cells bearing DNA damage was even higher in the model group. Besides, the change trends of the percentage of cells bearing DNA damage were different between the two groups (F =89.290, P = 0.000). The relative protein expression of γH2AX in the model group was higher than that in the control group (P < 0.05). The comparison of the percentage of >3 γH2AX-positive lesions under the fluorescence microscope on the 3rd and 12th day in the control group and the model group showed that the percentage of > 3 γH2AX-positive lesions under the fluorescence microscope was different between the time points (F =168.970, P =0.000) and between the groups (F =203.040, P = =0.000), in which the percentage of > 3 γH2AX-positive lesions under the fluorescence microscope was even higher in the model group that suggested obvious DNA damage. The change trends of the percentage of > 3 γH2AX-positive lesions under the fluorescence microscope were different between the model group and the control group (F = 153.410, P = 0.000). The level of 8-OHDG in the model group was higher than that of the control group (P < 0.05). The cell viability in the model group was lower than that in the iATM group and the iPARP group (P < 0.05), while the cell viability in the control group was not different from that in the iATM group and the iPARP group (P > 0.05).Conclusions High calcium and phosphorus may activate the DNA damage response signaling pathway and induce necrosis of HVSMC which in turn causes HVSMC calcification.