Toll-like receptors (TLRs) recognize specific microbial products in the innate immune response. TLR3, a double-stranded RNA sensor, is thought to have an important role in viral infections, but the regulation of TLR3 expression and its function in keratinocytes are not fully understood. Here we show the Th1 cytokine IFN-gamma increased the TLR3 expression via STAT1 in cultured normal human epidermal keratinocytes (NHEKs). Co-stimulation with IFN-gamma and the TLR3 ligand poly (I:C) synergistically increased the expression of IFN-beta, IL-6, IL-8, and human beta-defensin-2 in NHEKs compared with poly (I:C) or IFN-gamma alone. These synergistic inductions were significantly inhibited by an endosomal acidification inhibitor, chloroquine, and by TLR3 siRNA. Co-stimulation with IFN-gamma and poly (I:C) also significantly enhanced the anti-viral activity against herpes simplex virus type-1 in NHEKs compared with poly (I:C) or IFN-gamma alone. In addition to the in vitro findings, an immunohistochemical analysis revealed IFN-gamma positive cells surrounding herpetic vesicles. These findings indicate that IFN-gamma might contribute to the innate immune response to cutaneous viral infections by enhancing TLR3 expression and function in keratinocytes.