Influenza A virus (IAV), a deadly zoonotic pathogen, occasionally cross-species transmission among humans, swine and avian. The ectodomain of matrix protein 2 (M2e) is highly conserved in IAV and has been widely concerned in the development of universal influenza vaccines. Due to low immunogenicity, multi-copy M2e are usually displayed on the surface of nanoparticles to constitute universal nanovaccines. Here, we report that the permutation of the M2e affects the immune effect of the nanovaccine. Three M2e derived from humans, swine and avian IAV were inserted into the C-terminal of the Cap protein of porcine circovirus type 2 (PCV2) to form self-assembled nanovaccine. Immunoprotective effects of different M2e arrangements were explored in mice. Results showed that the M2e closest to the surface of nanoparticle induced the most efficient protection against IAV derived from corresponding species. The results will help in the development of more effective universal influenza vaccines, especially for specific species.