Histone lysine lactylation (Kla) has recently been reported to participate in various biological processes, regulating transcription, inflammation, and immune-related diseases. However, the mechanism of histone Kla in innate immunity and viral infection remains largely unknown. Here, we observed fluorescent Kla signals in all four histones (H2A, H2B, H3, and H4) in PK-15 cells. Immunoprecipitation analysis showed prominent histone Kla protein bands, with H2B being the most abundant. We generated the H2B K16R mutant plasmid and identified K16 as one of the Kla modification sites in H2B. Further exploration revealed increased global H2B Kla and H2BK16la levels upon classical swine fever virus (CSFV) infection. By employing the Kla agonist (L-lactate), inhibitor (oxamate), or siLDHA, we demonstrated that H2BK16la and pan Kla in PK-15 cells rely on the LDHA-lactate axis, which is also crucial for CSFV-induced H2BK16la and pan Kla levels. Moreover, our data proved the interaction between H2B and CSFV NS4A protein. Notably, H2B Kla can modulate CSFV proliferation. Mechanistically, H2BK16la and pan Kla activate the nuclear factor kappa B (NF-κB) pathway by mediating p65 nuclear translocation via karyopherin α2 (KPNA2), thereby inducing type III interferon (IFN-λ) expression and inhibiting CSFV replication. In conclusion, our study unveils the role of H2B Kla in regulating the NF-κB pathway during viral infection, presenting a novel mechanism. These findings significantly contribute to understanding the pathogenic mechanisms during viral infection and hold promise for the development of viral therapeutic strategies.