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 multi-copy M2e from different species are usually displayed on the surface of nanoparticles to improve immunogenicity and constitute universal IAV nanovaccines. In our previous study, three M2e were inserted into the C-terminal of Cap protein of porcine circovirus type 2 (PCV2) to form a universal nanovaccine that protects PCV2 and different subtypes of IAV. Howerer, M2e adopts at least two converted conformations, and the intermolecular linker of M2e enhances the conformational instability, which limits the recognition of B cell receptors and production of high-level antibodies. Here, we report that the permutation of the M2e affects immune effect of nanovaccines. Three M2e derived from humans, swine and avian IAV were inserted into the C-terminal of the Cap protein to form nanovaccines. 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 to develop more effective universal IAV and PCV2 bivalent nano-vaccines, as well as universal IAV vaccines for specific species.