The purpose of this study was to construct a Coxsackie virus A16 (CA16) mucosal vaccine and evaluate its ability to induce immune response. VP1 gene of CA16 was inserted into the genome of Bacillus subtilis via recombination and displayed on the surface of the spores. This Bacillus-based vaccine was used for intranasal immunization of mice and the serum antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). Neutralization activity of the serum from immunized mice was analyzed by an in vitro neutralizing test. VP1 gene was successfully integrated into the genome of Bacillus subtilis and was expressed on the surface of Bacillus spores. Intranasal immunization of mice with this vaccine induced a higher level of VP1 specific IgA and IgG than in mice of the control group (p < 0.05). The neutralizing antibody titer in the spore immunization group was 1: 169, which was higher than that in the control group (p < 0.05). We concluded that vaccine prepared by displaying CA16 VP1 protein on the surface of Bacillus subtilis spores can stimulate mice to produce protective neutralizing antibodies, which provides foundations for the development of CA16 mucosal vaccine.