The emergence of novel influenza A H1N1 and highly pathogenic avian influenza (HPAI) H5N1 viruses underscores the urgency of developing efficient vaccines against an imminent pandemic. M(NLS-88R) (H1N1), an A/WSN/33 mutant with modifications in the multibasic motif 101RKLKR105 of the matrix (M1) protein and its adjacent region, was generated by reverse genetics. The M(NLS-88R) mutant had in vitro growth characteristics similar to those of wild-type A/WSN/33 (wt-WSN), but it was attenuated in mice. Vaccination with M(NLS-88R) not only fully protected mice from lethal homologous challenges but also prevented mortality caused by antigenically distinct H3N2 and H5N1 viruses. M(NLS-88R)-induced homologous protection was mainly antibody dependent, but cellular immunity was also beneficial in protecting against sublethal wt-WSN infection. Adoptive transfer studies indicated that both humoral and cellular immune responses were crucial for M(NLS-88R)-induced heterologous protection. Our study suggests an alternative approach to attenuate wt influenza viruses for the development of a pandemic vaccine with broad cross-protection.