Double-stranded RNA (dsRNA) and its mimic, polyinosinic acid: polycytidylic acid [poly (I): poly (C)], are recognized by toll-like receptor 3 (TLR3) that induces the production of IFN-β in many cell types. In the present study, we investigated the effects of poly (I): poly (C) on mouse osteoblastic MC3T3-E1 (E1) cells. Poly (I): poly (C) markedly increased IFN-β mRNA level in a dose-dependent manner. The increase in the IFN-β mRNA level was apparent as early as 1 h after adding poly (I): poly (C) to the culture and peaked at 12 h. Stimulation with poly (I): poly (C) enhanced the expression of CXCL10 mRNA and TLR3 in E1 cells. Moreover, poly (I): poly (C) induced tyrosine phosphorylation of the transcription factor STAT1 in E1 cells. An anti-IFN-β neutralizing antibody partially inhibited poly (I): poly (C)-induced CXCL10 mRNA, TLR3 mRNA and STAT1 phosphorylation. These results indicate that osteoblasts secrete IFN-β in response to viral infection and that endogenous IFN-β induces both CXCL10 and TLR3 production via an IFN-α/β receptor-STAT1 pathway. It is suggested that osteoblasts are involved in host defense as well as bone metabolism.