To explore the effect of MDC1 gene silencing by RNA interference on the radiosensitivity of human esophageal carcinoma cell line ECA109.The vectors containing short hairpin RNA (shRNA) targeting MDC1 gene (pMDC1-shRNA) were cotransfected with pPACKH1-lentivector packaging system into 293T cells to package the lentivirus particles. Forty-eight hours after the transfection with specific or control lentiviral vectors, the stable integrants were selected using copGFP reporter gene; real-time RT-PCR and Western blotting were used to detect the expression levels of MDC1 mRNA and protein in the transfected ECA109 cells, respectively. The cell cycle distribution was measured with flow cytometry at 12, 24 and 48 h after a 5 Gy irradiation, and the radiosensitivity of esophageal carcinoma cell was evaluated by clone formation array.Sequence analysis confirmed correct insertion of MDC1-shRNA construct into pSIH1-H1-copGFP. The percentage of G2/M phase ECA109/ MDC1 cells was lower than that of ECA109 and ECA109/negative cells. The value of D0, SF2 and Dq of ECA109/ MDC1 cells were 1.88 Gy, 0.84 and 1.20, respectively, lower than those of ECA109 cells (3.06 Gy, 0.91 and 1.59) and those of ECA109/negative cells (2.90 Gy, 0.89 and 1.47).RNA interference can inhibit MDC1 gene expression and enhance the radiosensitivity of ECA109 cells in vitro.