Nuclear localization of cytoplasmic RNA virus proteins mediated by intrinsic nuclear localization signal (NLS) plays essential roles in successful virus replication. We previously reported that NLS mutation in the matrix (M) protein obviously attenuates the replication and pathogenicity of Newcastle disease virus (NDV), but the attenuated replication mechanism remains unclear. In this study, we showed that M/NLS mutation not only disrupted M's nucleocytoplasmic trafficking characteristic but also impaired viral RNA synthesis and transcription. Using TMT-based quantitative proteomics analysis of BSR-T7/5 cells infected with the parental NDV rSS1GFP and the mutant NDV rSS1GFP-M/NLSm harboring M/NLS mutation, we found that rSS1GFP infection stimulated much greater quantities and more expression changes of differentially expressed proteins involved in host cell transcription, ribosomal structure, posttranslational modification, and intracellular trafficking than rSS1GFP-M/NLSm infection. Further in-depth analysis revealed that the dominant nuclear accumulation of M protein inhibited host cell transcription, RNA processing and modification, protein synthesis, posttranscriptional modification and transport; and this kind of inhibition could be weakened when most of M protein was confined outside the nucleus. More importantly, we found that the function of M protein in the cytoplasm effected the inhibition of TIFA expression in a dose-dependent manner, and promoted NDV replication by down-regulating TIFA/TRAF6/NF-κB-mediated production of cytokines. It was the first report about the involvement of M protein in NDV immune evasion. Taken together, our findings demonstrate that NDV replication is closely related to the nucleocytoplasmic trafficking of M protein, which accelerates our understanding of the molecular functions of NDV M protein.