Anti-inflammatory agent 35 reduces ASFV replication by suppressing the nuclear translocation of p65

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly contagious infectious disease. To date, no safe and effective vaccine or therapeutic drug is available for ASFV. In this study, we identified Anti-inflammatory agent 35 (A35) as a negative regulator of ASFV replication. Real-time quantitative PCR, hemadsorption assay, and Western blotting analyses revealed that A35 inhibits ASFV genome replication and structural protein expression in vitro. Further studies demonstrated that A35 suppresses TNF-α or ASFV-triggered NF-κB signaling pathway. Reporter assays showed that A35 inhibited p65-triggered NF-κB signaling pathway. Mechanistic studies have found that A35 can inhibit the nuclear translocation of p65. Additionally, overexpression of p65 enhanced ASFV replication, whereas knockdown of p65 had opposite effects. This study reveals, for the first time, that A35 exerts its anti-ASFV effects by targeting NF-κB signal transduction, providing a theoretical basis and candidate compound for the development of novel anti-ASFV agents based on the structural optimization of natural products.