Atopic dermatitis (AD) is characterized by epidermal barrier defects and recurrent microbial skin infections. AD patients with a history of eczema herpeticum (ADEH+) have more severe skin disease and more highly T helper type 2 (Th2)–polarized immune responses as compared with uncomplicated AD (ADEH-). However, the mechanisms linking epidermal barrier defects and viral skin infection are not well understood. Recently, it has been reported that interleukin-25 may play a role in augmenting Th2 responses. We examined protein expression of IL-25 in the skin biopsies from normal subjects (n=10), ADEH- (n=18), ADEH+ (n=7), and psoriasis (n=9). IL-25 expression was increased in the skin from ADEH-, ADEH+, and psoriasis as compared with normal skin, and was significantly greater in lesional ADEH+ skin than in lesional ADEH- skin. Importantly, we demonstrated that IL-25 enhances herpes simplex virus (HSV)-1 and vaccinia virus replication by inhibiting filaggrin expression, and IL-25 acts synergistically with IL-4 and IL-13 to enhance HSV-1 replication in vitro. In contrast, IFN-γ inhibited HSV-1 replication in vitro. In addition, we demonstrate that filaggrin is a critical protein to inhibit HSV-1 replication because filaggrin small interfering RNA knockdown enhances HSV-1 replication in vitro. Filaggrin breakdown products, however, inhibited HSV-1 replication in vitro.