The matrix (M) protein of Newcastle disease virus (NDV) is a highly conserved hydrophobic viral protein. In some paramyxoviruses (measles virus and Sendai virus), the paired glycine (G) near the C terminus of the M protein may form a turn that mediates the specific interaction with the cell membrane. Similar amino acids (glycine-proline [GP], at position 275-276) exist in the M protein of NDV. However, the role of these residues in the replication and pathogenicity of NDV is unknown. In this study, recombinant NDV with the sequence GP/AA or LGP/GGL in the M protein was generated to investigate the role of this conserved sequence. Budding experiments on the mutant viruses revealed that the GP/AA mutation reduced virus budding and virus replication in DF-1 cells; biological characterization revealed attenuated virulence and pathogenicity in chickens, indicating that the GP sequence plays a critical role in the life cycle of the virus.