Viral infections can disturb the functions of adipose tissues and thus result in metabolic diseases. Polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analog of viral double-stranded RNA, induces innate antiviral responses by mimicking viral infection through the activation of pattern recognition receptors (PRRs) such as Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Poly(I:C) also inhibits the differentiation of mouse preadipocytes but the mechanism underlying this process remains unclear. In this study, poly(I:C) inhibited preadipocyte differentiation in a dose-dependent manner, but not in a time-dependent manner. Endogenously transfected poly(I:C) severely impaired the adipogenesis of preadipocytes compared with exogenously added poly(I:C). Low concentration of tumor necrosis factor-α (TNF-α) could effectively inhibit the preadipocyte differentiation. The effect of exogenously added poly(I:C) on inhibition of differentiation was significantly diminished in the preadipocytes of TLR3 knockout mice. By contrast, endogenously transfected poly(I:C) still inhibited the differentiation of TLR3-deficient preadipocytes. Hence, MDA5/RIG-I signaling was involved in the poly(I:C)-induced inhibition of preadipocyte differentiation. The effect of poly(I:C) stimulation, either through endogenous transfection or exogenous addition, on inhibition of differentiation was significantly diminished in the preadipocytes of TNF-α knockout mice. These results confirmed the evidence that poly(I:C) inhibited the differentiation of mouse preadipocytes through PRR-mediated secretion of TNF-α.