Objective Coxsackie virus group B type 5 (CVB5) is one of the causative agents of hand-foot- mouth disease, which can cause clinical symptoms such as fever, rash or herpes, and neurological complications or even fatalities. The innate immune response is the first line of defense against the viral infection, and the nuclear factor-κB (NF-κB) is a master regulator in the control of immune responses. However, little research has been reported on the regulation of the NF-κB mediated signaling pathway after CVB5 infection. This study explores the regulatory mechanism of virus and the host innate immune response, providing targets for the development of drugs against CVB5 infection.Methods In this study, promoter activity, proinflammatory factor and key proteins expression were detected to investigate the regulatory mechanism of CVB5 on NF-κB signaling.Results CVB5 infection inhibited the expression of proinflammatory factors and the phosphorylated p65 protein expression. Non-structural protein (NSP) of CVB5 inhibited the expression of proinflammatory factor and important proteins, such as the phosphorylated p65 and IκBα. CVB5 3CD interacted with the host polycytosine binding protein 1 (PCBP1) was performed via the STRING 11.1 database, and the PCBP1 inhibited viral replication by promoting the phosphorylation of IκBα and p65.Conclusion These results showed that CVB5 NSP negatively regulated NF-κB signaling pathway, and the PCBP1 protein which interacted with 3CD could inhibit CVB5 replication through activate the NF-κB pathway.