Viral respiratory tract infections are the most common illness in humans. Infection of the respiratory viruses results in accumulation of viral replicative double-stranded RNA (dsRNA), which is one of the important components of infecting viruses for the induction of lung epithelial cell apoptosis and innate immune response, including the production of interferon (IFN). In the present study, we have investigated the regulation of dsRNA-induced airway epithelial cell apoptosis by IFN. We found that transcription factor Runx3 was strongly induced by type-II IFN, slightly by type-III IFN, but essentially not by type-I IFNα in airway epithelial cells. IFN-induced expression of Runx3 was predominantly mediated by JAK-STAT1 pathway and partially by NF-B pathway. Interestingly, Runx3 can be synergistically induced by IFN with a synthetic analog of viral dsRNA polyinosinic-polycytidylic acid (poly(I:C)) or tumor necrosis factor-α (TNFα) through both JAK-STAT1 and NF-B pathways. We further found that dsRNA poly(I:C)-induced apoptosis of airway epithelial cells was mediated by dsRNA receptor Toll-like receptor 3 (TLR3) and was markedly augmented by IFN through the enhanced expression of TLR3 and subsequent activation of both extrinsic and intrinsic apoptosis pathways. Last, we demonstrated that upregulation of Runx3 by IFN promoted TLR3 expression, thus amplifying the dsRNA-induced apoptosis in airway epithelial cells. These novel findings indicate that IFN promotes dsRNA-induced TLR3-dependent apoptosis via upregulation of transcription factor Runx3 in airway epithelial cells. Findings from our study may provide new insights into the regulation of airway epithelial cell apoptosis by IFN during viral respiratory tract infection.