Active virus-host interactions determine the outcome of pathogen invasions. It has been shown that in isolated dendritic cells (DCs), rotavirus can induce the expression of tumor necrosis factor α (TNF-α), a vital cytokine mediating host immune responses. However, the role of TNF-α in rotavirus infection is unknown. In this study, we demonstrated that TNF-α has potent anti-rotavirus effects, independent of type I interferon production. Blocking of TNF-α by infliximab, a clinically available TNFα antibody, totally abrogated this effect. Mechanistic studies revealed that the anti-rotavirus effect of TNF-α was achieved by NFκB-regulated genes via the activation of classical nuclear factor κB (NF-κB) signaling. Our study reveals the pivotal role and the mechanism-of-actions of TNF-α in the host defense against rotavirus. Thus, this knowledge may contribute to the better understanding of the complexity of rotavirus-host interactions.