BX-795 is an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1), but also a potent inhibitor of the IKK-related kinase, TANKbinding kinase 1 (TBK1) and IKKɛ. In this study we attempted to elucidate the molecular mechanism(s) underlying the inhibition of BX-795 on Herpes simplex virus (HSV) replication. HEC-1-A or Vero cells were treated with BX-795 and infected with HSV-1 or HSV-2 for different periods. BX-795 (3.125-25 µmol/L) dose-dependently suppressed HSV-2 replication, and displayed a low cytotoxicity to the host cells. BX-795 treatment dose-dependently suppressed the expression of two HSV immediate-early (IE) genes (ICP0 and ICP27) and the late gene (gD) at 12 h postinfection. HSV-2 infection resulted in the activation of PI3K and Akt in the host cells, and BX-795 treatment inhibited HSV-2-induced Akt phosphorylation and activation. However, the blockage of PI3K/Akt/mTOR with LY294002 and rapamycin did not affect HSV-2 replication. HSV-2 infection increased the phosphorylation of JNK and p38, and reduced ERK phosphorylation at 8 h postinfection in the host cells; BX-795 treatment inhibited HSV-2-induced activation of JNK and p38 MAP kinase as well as the phosphorylation of c-Jun and ATF-2, the downstream targets of JNK and p38 MAP kinase. Furthermore, SB203580 (a p38 inhibitor) or SP600125 (a JNK inhibitor) dose-dependently inhibited the viral replication in the host cells, whereas PD98059 (an ERK inhibitor) was not effective. Moreover, BX-795 blocked PMA-stimulated c-Jun activation as well as HSV-2-mediated c-Jun nuclear translocation. BX-795 dose-dependently inhibited HSV-2, PMA, TNF-α-stimulated AP-1 activation, but not HSV-induced NF-κB activation. Overexpression of p38/JNK attenuated the inhibitory effect of BX-795 on HSV replication. BX-795 completely blocked HSV-2-induced MKK4 phosphorylation, suggesting that BX-795 acting upstream of JNK and p38 MAP kinase. In conclusion, this study identifies the anti-HSV activity of BX-795 and its targeting of the JNK/p38 MAP kinase pathways in host cells.