Emerging evidence indicates that methyltransferase NSUN2 catalyzes 5-methylcytosine (m5C) modifications on various viral RNAs and plays important roles in viral biology. However, the regulatory roles of NSUN2 in influenza A virus (IAV) replication have not been elucidated. Here, we revealed that NSUN2 negatively regulated the viral RNA transcription and protein production by knocking out and over-expressing NSUN2. By m5C MeRIP-seq and RNA-BisSeq, NSUN2-dependent m5C sites were identified on both the plus and minus viral RNA strands. In NSUN2-KO cells, the m5C modification on vRNAs was reduced, resulting in the production of a large number of deficient interfering particles (DIPs) which had lower vRNA content, imbalanced genome fragments, abnormal morphology and reduced pathogenicity. Mutation of m5C sites at the 5´ and 3´ ends of PB2 vRNA interfered with the selective packaging of the 8 vRNA segments into virus particles, resulting in the formation of a variety of abnormally packaged virus particles. PB2-vRNA mutants also had reduced replication ability and pathogenicity in mice. Overall, these data demonstrate that the m5C residues catalyzed by NSUN2 are required on vRNAs for the proper assembly of infectious viral particles, suggesting the depletion of m5C modification as a potential strategy that can be utilized to attenuate IAV strains.