Spike (S) protein is an essential transmembrane glycoprotein on the surface of coronaviruses. It has dual functions of receptor binding and membrane fusion for infection. Although most of the receptor binding domains (RBD) of S proteins are located in S1-CTD, or domain B, the receptor for beta-coronavirus mouse hepatitis virus (MHV) binds to S1-NTD, or domain A, and the role of domain B remains unclear. By constructing and expression of domain B deletion mutants of S protein with S2 membrane fusion in neuroblastoma cell line Neuro-2a, we proved that the domain B is essential for cell-cell membrane fusion mediated by the S protein. Further analysis of polyclonal antibodies generated in mice revealed that domain B is not only the major antigenic determinant of S protein, but also a domain for significant neutralizing antibodies to inhibit viral infection. These results provided insights into the role of S protein in pathogenicity and infection of coronaviruses.