Herpes simplex virus type 1 (HSV-1) is human specific virus. The intercellular transmission of HSV-1 is essential in its pathogenesis. The tunneling nanotube (TNT), a new mode connecting distant cells, has been found to play an important role in the spread of various viruses like human immunodeficiency virus (HIV) and influenza virus. However, whether HSV-1 can be transmitted through TNTs has not been confirmed. The purpose of this study was to clarify this, and further to determine the effect of inhibiting the actin-related protein 2/3 (Arp2/3) complex on the intercellular transmission of HSV-1. A scanning electron microscope and fluorescence microscope detected the formation of TNTs between HSV-1 infected cells. Envelope glycoprotein D (gD) and envelope glycoprotein E (gE) of HSV-1 and viral particles were observed in TNTs. Treatment with CK666, an inhibitor of the Arp2/3 complex, reduced the number of TNTs by approximately 40-80%. At the same time, the DNA level of HSV-1 in cells and the number of plaque formation units (PFU) were also reduced by nearly 30%. These findings indicated that TNT contributes to HSV-1 transmission and that the inhibition of the Arp2/3 complex could impair HSV-1 transmission, which not only provides a novel insight into the transmission mode of HSV-1, but also a putative new antiviral target.