Objectives: Herpes simplex virus 1 (HSV-1) is one of the most prevalent viruses in humans worldwide. Owing to limited therapeutic options mainly with acyclovir (ACV) and analogues and the emergence of ACV-resistant strains, new drugs with different modes of action and low toxicity are required. The aim of this study was to determine the anti-HSV-1 effect and mechanism of action of the flavonoid compound dihydromyricetin (DHM) from Ampelopsis grossedentata. Methods: The HSV-1 inhibitory effect of DHM was evaluated by measuring plaque formation and generation of progeny virus as well as expression of HSV-1-related genes in Vero cells. The molecular mechanism of the antiviral activity of DHM against HSV-1 was explored by real-time quantitative PCR and ELISA. Results: DHM presented a significant inhibitory effect on HSV-1 plaque formation and generation of progeny virus, with an EC50 (50% effective concentration) of 12.56 mu M in Vero cells. Furthermore, expression of HSV-1 immediate-early genes (ICP4 and ICP22), early genes (ICP8 and UL42) and late genes (gB, VP1/2) was decreased by DHM at concentrations of 16 mu M and 32 mu M. DHM specifically suppressed mRNA levels of Toll-like receptor 9 (TLR9), leading to inhibition of the inflammatory transcriptional factor NF kappa B and a decrease in THF alpha. Conclusion: These findings indicate that the effective inhibitory activity of DHM was achieved by suppressing TNF alpha production in a TLR9-dependent manner. Although further studies are needed to better characterise the activity of DHM in vivo, the results suggest this extract as a promising new anti-HSV-1 agent. (C) 2020 The Authors. Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.