Exposure of human monocytic cells to herpes simplex virus type 1 (HSV-1) results in immediate up-regulation of interleukin (IL)-15 gene expression. However, the receptor involved in this induction is not known. Here, we provide evidence that this induction depends on TLR2-mediated signaling pathway. Through the use of small interfering RNAs (siRNAs), we demonstrate that HSV-1-induced upregulation of IL-15 gene expression in monocytic THP1 cells requires the presence of the adaptors MyD88, IRAKI, and TRAF6. Interestingly, TIRAP/Mal, an adaptor molecule specifically recruited to TLR2 and TLR4, was also required for maximal up-regulation of IL-15. This response was completely abrogated by anti-TLR2, but not anti-TLR4, blocking mAbs in both primary monocytes and THP1 cells. Furthermore, THP1 cells rendered defective in TLR2 expression by disrupting the expression of Sp1, a major transcription factor involved in TLR2 promoter activity, were unable to up-regulate IL-15 gene expression in response to HSV-1. In addition, HSV-1-induced NF-κ B activation was significantly reduced after neutralization of TLR2 and the adaptor proteins. Altogether, these results unequivocally show that HSV-1 induces TLR2-dependent activation of IL-15 gene expression, which requires the recruitment of both MyD88 and TIRAP/Mal and the activation of IRAKI and TRAF6 leading to NFκB translocation to the nucleus.