〔Abstract〕 Objective To investigate the inhibitory effect and molecular mechanism of ribosomal translation factor inhibitor Avilamycin on hepatitis B virus replication. Methods Liver cancer Hep3 B cells were treated with different concentrations of Avilamycin. Cell activity was detected by CCK-8; the apoptosis was detected by flow cytometry, and HBV-DNA、pgRNA、MTIF2、RPL10 gene expression level was detected by qPCR method. The HBsAg and HBeAg was detected by ELISA. The AFP was detected by chemiluminescence. Aspartate aminotransferase(AST), alanine aminotransferase(ALT), and alkaline phosphatase(ALP) proteins was detected by Biochemistry method. Results Avilamycin had no inhibitory effect on Hep3 B cell proliferation and apoptosis. However, it could promote cellular AST secretion, reduce AFP levels, and have less effect on ALP secretion. In Hep3 B cells, Avilamycin promotes accumulation of pgRNA expression by intervening with MTIF2 and feedback upregulates mRNA expression of host RPL10 and MTIF2 genes. It can effectively reduce the HBsAg, HBeAg, and HBV-DNA levels. Conclusion Avilamycin can inhibit MTIF2 translation initiation, regulate the translation process of viral assembly protein by affecting translation initiation, and then inhibit hepatitis B virus replication.