The essential contribution of mast cells (MCs) to bacterial host defense has been well established; however, little is known about their role in viral infections in vivo. Here, we found that intradermal injection with herpes simplex virus 2 (HSV-2) into MC-deficient Kit(W/Wv) mice led to increased clinical severity and mortality with elevated virus titers in HSV-infected skins. Ex vivo HSV-specific tetramer staining assay demonstrated that MC deficiency did not affect the frequency of HSV-specific cytotoxic T lymphocytes (CTLs) in draining lymph nodes. Moreover, the high mortality in Kit(W/W-v) mice was completely reversed by intradermal reconstitution with bone marrow-derived MCs (BMMCs) from wild-type, but not TNF(-/-) or IL-6(-/-) mice, indicating that MCs or, more specifically, MC-derived tumor necrosis factor (TNF) and IL-6 can protect mice from HSV-induced mortality. However, HSV did not directly induce TNF-α or IL-6 production by BMMCs; supernatants from HSV-infected keratinocytes induced the production of these cytokines by BMMCs without degranulation. Furthermore, IL-33 expression was induced in HSV-infected keratinocytes, and blocking the IL-33 receptor T1/ST2 on BMMCs significantly reduced TNF-α and IL-6 production by BMMCs. These results indicate the involvement of MCs in host defense at HSV-infected sites through TNF-α and IL-6 production, which is induced by keratinocyte-derived IL-33.