Swine influenza A virus (SIV) is both a pathogen of economic significance to the swine industry and a potential zoonotic organism that may be transmitted to humans. We described here the detailed characterization of a role of N-terminal B-loop and CD helix of HA2 in swine influenza A virus replication. Results of our experiments demonstrated that Hemagglutinin (HA) protein of swine influenza virus could tolerate some mutations in functionally conserved B-loop and CD helix. These mutations, however, have substantially attenuated influenza virus replication in both cell lines and porcine primary tracheal epithelial cells. Significantly, we found that some B-loop or CD helix mutations generated virus mutants that replicated in MDCK and ST cell lines but failed to replicate in primary tracheal epithelial cells, thereby suggesting that swine HA protein may function as a viral virulence and pathogenesis factor. The described mutations may be further explored as attenuated vaccine candidates that can effectively prevent or eliminate the spread of influenza virus within and between swine herds.