could inhibit HSV-2 infection, with 50% inhibitory concentrations of 5.58 µM and 6.35 µM in cytopathic effect inhibition and plaque reduction assays, respectively. The cytotoxicity of Retro-2.1 was relatively low, with a 50% cytotoxicity concentration of 116.5 µM.We also preliminarily identified that Retro-2.1 exerted the antiviral effect against HSV-2 by a dual mechanism of action on virus entry and late stages of infection. Therefore, our study for the first time demonstrated Retro-2.1 as an effective antiviral agent against HSV-2 in vitro with targets distinct from those of nucleoside analogs.Keywords: Herpes simplex virus type 2, Retro-2.1, antiviral effect, entry, late stage, vesicle transport 850 Dai et al. J. Microbiol. Biotechnol.