ABSTRACTAfrican swine fever (ASF), caused by the African swine fever virus (ASFV), is a transboundary infectious disease of domestic pigs and wild boars, resulting in significant swine production losses. Currently, no effective commercial ASF vaccines or therapeutic options are available. A previous study has shown that deletions of ASFVMGF110-9LandMGF505-7Rgenes (ASFV-Δ110-9L/505-7R) attenuated virulence in pigs and provided complete protection against parental lethal ASFV CN/GS/2018 (wild-type ASFV [ASFV-WT]) challenge, but the underlying mechanism is unclear. This study found that ASFV-Δ110-9L/505-7R weakened TBK1 degradation compared with ASFV-WT through RNA sequencing (RNA-seq) and Western blotting analyses. Furthermore, we confirmed that ASFV-Δ110-9L/505-7R blocked the degradation of TBK1 through the autophagy pathway. We also identified that the downregulation of an autophagy-related protein PIK3C2B was involved in the inhibition of TBK1 degradation induced by ASFV-Δ110-9L/505-7R. Additionally, we also confirmed that PIK3C2B promoted ASFV-Δ110-9L/505-7R replicationin vitro. Together, this study elucidated a novel mechanism of virulence change of ASFV-Δ110-9L/505-7R, revealing a new mechanism of ASF live attenuated vaccines (LAVs) and providing theoretical guidance for the development of ASF vaccines.IMPORTANCEAfrican swine fever (ASF) is a contagious and lethal hemorrhagic disease of pigs caused by the African swine fever virus (ASFV), leading to significant economic consequences for the global pig industry. The development of an effective and safe ASF vaccine has been unsuccessful. Previous studies have shown that live attenuated vaccines (LAVs) of ASFV are the most effective vaccine candidates to prevent ASF. Understanding the host responses caused by LAVs of ASFV is important in optimizing vaccine design and diversifying the resources available to control ASF. Recently, our laboratory found that the live attenuated ASFV-Δ110-9L/505-7R provided complete protection against parental ASFV-WT challenge. This study further demonstrated that ASFV-Δ110-9L/505-7R inhibits TBK1 degradation mediated by an autophagy activator PIK3C2B to increase type I interferon production. These results revealed an important mechanism for candidate vaccine ASFV-Δ110-9L/505-7R, providing strategies for exploring the virulence of multigene-deleted live attenuated ASFV strains and the development of vaccines.