Double-stranded (ds) RNA has been identified as a ligand for Toll-like receptor 3 (TLR3). Respiratory syncytial virus (RSV), a single-stranded RNA virus and a major respiratory pathogen and pneumovirus in human infants pathogenesis of which relies on early inflammatory and immune events of the host in response to RSV, could be recognized by TLR3 sensing viral dsRNA produced during replication. The downstream signaling pathway from TLR3 leads to activation of IFN regulatory factor (IRF)-3 and/or NF-kappaB and subsequent expression of numerous proinflammatory factors. Melatonin (MT) is an effective regulator of the immune system. To determine the molecular mechanisms responsible for the suppressive effect of MT on RSV infection, we analyzed signaling molecules involved in the TLR3-mediated activation of inflammatory factors in macrophages infected with RSV and the modulatory role of MT on these mediators. We report that RSV infection of RAW264.7 macrophages time-dependently stimulate the rapid activation of TLR3 and NF-kappaB, as well as subsequent NF-kappaB-dependent gene expression such as those encoding TNF-alpha and inducible nitric oxide synthase. Moreover, we demonstrate that MT decreased TLR3-mediated downstream gene expression in RSV-infected macrophages in a dose- and time-dependent manner, and that MT inhibition of NF-kappaB activity seemed to be the key event required to explain the reduction in inflammatory gene expression caused by MT. But MT did not influence TLR3 at either the protein or mRNA level or MyD88 transcription. These results could be related to the beneficial immunoregulatory role of MT in RSV-infected macrophages and address the possible therapeutic potential of this indoleamine in human RSV diseases.