Coxackievirus A16 (CA16) is a major pathogen of hand, foot and mouth disease. Because of the unclear pathogenesis and immunomechanism of CA16, the development of a CA16 vaccine has encountered numerous difficulties. Many studies have reported that epithelial cells and associated immune cells in respiratory tract mucosa, especially innate lymphoid cells (ILCs), are largely contributed to the activation of innate and adaptive immune responses during viral infection. It is still unknown that whether ILCs play a similar role in the immune response induced by CA16 infection. To reveal the potential interactions between ILCs and CA16 infection, the expression levels of genes related to ILC activation in CA16-infected human tracheal epithelial cells (16HBE) and mouse respiratory tract epithelial cells (CP-M175) stimulated by adjuvants formulated CA16-inactivated antigen were firstly determined. Then, a formulated experimental CA16-inactivated vaccine was used to immunize BALB/c mice through nasal route, and the relationship between ILCs and viral antigens in the respiratory mucosa was analyzed. Both CA16 live virus and inactivated virus particles could induce upregulation of various molecules required for the activation of ILCs and stimulate transcription and expression of cytokines, which were secreted by activated ILCs, no matter in vitro and in vivo. Moreover, immunofluorescence and confocal microscopic results showed that the viral antigen was co-localized with ILC1/ILC3. Finally, the anti-CA16 neutralizing antibodies and specific cellular immune response in mice of four different immunization pathways/immunization procedures were tested. The results suggested that the mice immunized with the formulated CA16-inactivated vaccine could induce an effective antiviral immune response in vivo. In conclusion, CA16 antigen can induce an effective immune response by activating ILCs.