Abstract: Objective To study the appropriate incubation and proliferation conditions of Herpes Simplex virus type 2（HSV-2） in Vero cells from African green monkeys. Methods HSV-2 was cultured and sub-cultured after inoculated into Vero cells.Virus suspension was harvested at different time points and titration of virus was tested.The proliferation condition of HSV-2 was investigated. Results The pH value and residual calf serum were the influencing factor for the proliferation of HSV-2 in Vero cells.The optimal time to harvest the virus was 36-72 hours after inoculation.The toxicity of HSV-2 was increased after 3-5 passages of HSV-2 in Vero cells and reached its peak after 10 passages. Conclusion The incubation and proliferation procedure for HSV-2 has been established,which lays a foundation for the establishment of HSV-2 latency and reactivation in vitro.