Ethnopharmacological relevance: The Chinese Herbal Prescription JieZe-1(JZ-1), added and subtracted from Yihuang Decoction, a famous formula in the 12th year of Kangxi in Qing Dynasty, has a clear effect on Genital Herpes (GH) and no obvious adverse reactions occur clinically. JZ-1 also has preventive and therapeutic effects on Trichomonas vaginitis, Candida albicans vaginitis and GH in vitro and in vivo experiments. Aim of study: The effect and mechanism of JZ-1 on anti-herpes simplex virus type 2(HSV-2) in vitro focusing on adhesion and penetration stages were investigated. Materials and methods: A model of HSV-2 infection of VK2/E6E7 was developed. In order to explore JZ-1's antiHSV-2 effect in vitro, cell morphology, ultrastructural pathology, cell viability and expression of viral glycoprotein D (gD) were assessed at 6 h, 12 h, 18 h, and 24 h of JZ-1 treatment. Then we measured the exact time required for adhesion and penetration of HSV-2 into VK2/E6E7 among a series of times at room temperature and under temperature control techniques. We treated VK2/E6E7 with JZ-1, penciclovir, or berberine and explored the mechanism of JZ-1 in blocking HSV-2 adhesion and penetration of host cells by assessing the cell ultrastructural pathology, viability, viral proteins gB, gD, VP16, ICP5, and ICP4 and host cell proteins HVEM, Nectin-1, and Nectin-2. Results: HSV-2 can fully adhere and penetrate into VK/E6E7 within 5 mins at room temperature while it takes 60mins under temperature control techniques. JZ-1 and penciclovir showed significant anti-HSV-2 effects, with improved host cell morphologies and increased host cell viabilities observed after treatment for 24 h. The antiHSV-2 effect of JZ-1 can be detected after treatment for 6 h while that of penciclovir was not obvious until treatment for 12 h. JZ-1 showed distinct effect on HSV-2 adhesion and penetration stages by significantly reducing the expression of viral proteins gB, gD, VP16, ICP5, and ICP4, improving cell morphology and increasing cell viability. However, these effects were not exerted via downregulated expression of membrane fusion-related proteins such as HVEM, Nectin-1, or Nectin-2. The specific anti-HSV-2 mechanism of JZ-1 need to be further explored. Conclusion: The anti-HSV-2 effect of JZ-1 was superior to that of penciclovir and berberine in vitro, and was mainly mediated by enhancing host cell defense and blocking adhesion and penetration of HSV-2.