Coxsackievirus B (CVB) represents one of the significant human pathogens that have been linked to several central nervous system disorders, particularly common among newborns and children. The annual outbreaks of CVB infection, pose a significant public health challenge and burden due to the absence of specific antiviral drugs and vaccines. However, the neuropathology of CVB infection remains elusive. The type I interferon response is well characterized for its role in controlling virus-induced neuropathogenesis. C19orf66 is known to be a potent interferon-stimulated gene with broad-spectrum antiviral activity, exerting its effects through diverse underlying molecular mechanisms. In this work, our study demonstrated that CVB induces the upregulation of C19orf66 both in host cells and in mice. Knockdown and overexpression of C19orf66 in CVB3-infected cells suggested that this factor could significantly suppress CVB3 replication. Our findings further revealed an intriguing mechanism by which C19orf66 could interact with the non-structural proteins 3D