[Objective] This study aimed to investigate the effect of nuclear localization signal (NLS) mutation in the M protein on the virulence and replication of duck-origin Newcastle disease virus (NDV). [Methods] The target fragment owning M/NLS mutation was obtained by overlap PCR method and then used to replace the corresponding region of pNDV/SS1GFP to construct pNDV/SS1GFP-M/NLSm. The M/NLS mutant virus rSS1GFP-M/NLSm was rescued by reverse genetics technology and identified by hemagglutination (HA) assay, fluorescence assay and sequencing analysis of the M gene. The subcellular localization of M protein, biological characteristics, plaque formation ability and proliferation ability of the rescued virus were also detected. [Results] The full-length plasmid pNDV/SS1GFP-M/NLSm was successfully constructed. The allantoic fluid obtained from the first generation had no HA titer, but the HA titer of the rescued virus was detected after three extra chicken egg passages. The results of fluorescence assay and M gene sequencing demonstrated that the rescued virus was rSS1GFP-M/NLSm. In comparison to the parental virus rSS1GFP, the M protein of rSS1GFP-M/NLSm mainly localized in the cytoplasm. In addition, the virulence, the replication ability and the plaque formation capacity of rSS1GFP-M/NLSm were obviously reduced. Moreover, the cytopathic effect and the expression of M protein and green fluorescent protein in rSS1GFP-M/NLSm-infected cells were also markedly decreased, indicating that the in vitro proliferation ability of duck-origin NDV was inhibited by M/NLS mutation. [Conclusion] The disruption of M's nuclear localization caused by M/NLS mutation could obviously attenuate the virulence and replication of duck-origin NDV.