Objective: HSV-2 infection has increased significantly in recent years, which is closely associated with cervical cancer and HIV infection. The lack of success in vaccine development and the emergence of drug resistance to commonly used drugs emphasize the urgent need for alternative antivirals against HSV-2 infection. Arbidol (ARB) has been demonstrated to be a broad spectrum antiviral drug that exhibits immunomodulatory properties that affect the HSV-2 life cycle. This study investigated the efficacy and mechanism of ARB against HSV-2 in vivo and in vitro to further explore the clinical application of ARB. Methods: The efficacy of ARB on HSV-2 infection in vitro was examined by CPE and MTT assays. A vaginitis model was established to monitor changes in histopathology and inflammatory cytokine (IL-2, IL-4, TNF-α and TGF-β) expression by H&E staining and ELISA, respectively, and the efficacy of ARB was evaluated accordingly. Furthermore, flow cytometry was used to determine the ratio of CD4 + /CD8 + T cells in the peripheral blood of the vaginitis animals. Considering the balance of efficacy and pharmacokinetics, ARB ointment was strictly prepared to observe formulation efficacy differences compared to the oral dosing form. Results: The results showed that, in vitro, the TC 50 and IC 50 of ARB were 32.32 μg/mL and 4.77 μg/mL (SI = 6.82), respectively, indicating that ARB presents effective activity against HSV-2 in a dose-dependent manner. The results of the time-course assay suggested that 25 μg/mL ARB affected the late stage of HSV-2 replication. However, ARB did not inhibit viral attachment or cell penetration. The in vivo results showed that ARB ointment can improve the survival rate, prolong the survival time and reduce the reproductive tract injury in mice infected with HSV-2, regulate cytokine expression; and balance the CD4 + and CD8 + T lymphocyte ratio in the peripheral blood to participate in the regulation of immune response. Conclusion: ARB showed anti-HSV-2 activity in vitro in a dose-dependent manner and played a role in inhibiting the late replication cycle of the virus. The vaginitis model was successfully established, according to immunomodulation outcomes, responded better to ARB in ointment form than in oral form. © 2018