Toll-like receptor 3 (TLR3) recognizes double-stranded RNA and induces type I interferon (IFN)-mediated antiviral immunity against a number of viral infections. Type III IFN (IFN-λ) is a newly identified antiviral cytokine that has biological functions similar to those of type I IFNs. We thus investigated the role of IFN-λ in TLR3 activation-mediated inhibition of herpes simplex virus type 1 (HSV-1) in human primary astrocytes. Human astrocytes express endogenous IFN-λ1 and IFN-λ receptor complex, interleukin-28 receptor α subunit (IL-28Rα), and IL-10Rβ. The activation of TLR3 by poly-I:C treatment significantly induced the expression of IFN-λ1 and IFN-λ2/3 in astrocytes. The induction of IFN-λ contributed to TLR3 activation-mediated HSV-1 inhibition in astrocytes. Investigation of the mechanisms showed that treatment of astrocytes with specific antibody against IFN-λ receptor attenuated the anti-HSV-1 activity of poly-I:C, indicating that endogenous IFN-λ contributes to the anti-HSV-1 effect of TLR3 activation. The anti-HSV-1 effect of endogenous IFN-λ was also confirmed by the finding that recombinant IFN-λ treatment inhibited HSV-1 infection of astrocytes. These results provide direct and compelling evidence that endogenous IFN-λ participates in TLR3-mediated antiviral activity, which may have important implications in host cell innate immunity against HSV-1 infection in the CNS.