Aim: To evaluate the efficiency of 3 short hairpin RNA (shRNA) interfering with the herpes simplex virus type l (HSV-I) gene coding glycoprotein D (gD) for inhibiting the gD expression and virus replication in vitro. Methods: Vero cells were selected for an in vitro model of infection. Three shRNA sequences (shRNA- gDl,-gD2, and-gD3) targeting specifically the gD gene of HSV-I were selected for evaluating the antiviral effects. The antiviral effects of shRNA in the cells in- fected with HSV-1 were evaluated by cytopathic effect (CPE) observations and plaque assays. The transcription level of viral RNA and the gD expression were studied by RT-PCR, Weste blotting, and flow cytometry. Results: With the 3 shRNA at a final concentration of 120 nmol/L, a significant inhibition of CPE in the HSV-l-infected cells was observed. The ED50 of shRNA-gDI, gD2, and gD3 were 48.74±2.57, 57.13±3.24, and 114.64±5.12 nmol/L, respectively. The gD gene decreased significantly after viral infection in the Vero cells pretreated with shRNA compared to the virus group. The expressions of the gD protein, determined by Weste blotting and flow cytometry, were also drastically decreased in shRNA-transfected cells. Conclusion: Exogenous shRNA molecules can suppress the HSV-1 gD expression. They are inhibitors of HSV replication during infection in Vero cells.