Viral infections can give rise to secondary bacterial infections. In the present study, we examined the role of invariant natural killer T (iNKT) cells in lipopolysaccharide (LPS)-induced lethal shock during encephalomyocarditis virus (EMCV) infection. Wild-type (WT) mice and Jα18 gene knockout (Jα18 KO) mice were inoculated with EMCV, 5days prior to challenging with LPS. The survival rate of Jα18 KO mice subjected to EMCV and LPS was significantly higher than that of WT mice. TNF-α and nitric oxide (NO) production were increased in WT mice, than that in Jα18 KO mice, after the administration of EMCV and LPS. EMCV infection increased the number of iNKT cells and IFN-γ production by iNKT cells in WT mice. Moreover, EMCV infection enhanced the expression of Toll-like receptor 4 (TLR4) in the lung and spleen. IFN-γ also increased the expression of TLR4 in splenocytes. These findings indicated that EMCV infection activated iNKT cells, and IFN-γ secreted from the iNKT cells up-regulated the expression of TLR4 in various tissues. As a result, EMCV-infected mice were susceptible to LPS and easily developed the lethal shock. In conclusion, iNKT cells were involved in the development of LPS-induced lethal shock during EMCV infection.