AbstractHepatitis B virus (HBV) infection is a major global health burden with 820 000 deaths per year. In our previous study, we found that the knockdown of autophagy‐related protein 5 (ATG5) significantly upregulated the interferon‐stimulated genes (ISGs) expression to exert the anti‐HCV effect. However, the regulation of ATG5 on HBV replication and its underlying mechanism remains unclear. In this study, we screened the altered expression of type I interferon (IFN‐I) pathway genes using RT² Profiler™ PCR array following ATG5 knock‐down and we found the bone marrow stromal cell antigen 2 (BST2) expression was significantly increased. We then verified the upregulation of BST2 by ATG5 knockdown using RT‐qPCR and found that the knockdown of ATG5 activated the Janus kinase/signal transducer and activator of transcription (JAK‐STAT) signaling pathway. ATG5 knockdown or BST2 overexpression decreased Hepatitis B core Antigen (HBcAg) protein, HBV DNA levels in cells and supernatants of HepAD38 and HBV‐infected NTCP‐HepG2. Knockdown of BST2 abrogated the anti‐HBV effect of ATG5 knockdown. Furthermore, we found that ATG5 interacted with BST2, and further formed a ternary complex together with HBV‐X (HBx). In conclusion, our finding indicates that ATG5 promotes HBV replication through decreasing BST2 expression and interacting with it directly to antagonize its antiviral function.