Hepatitis B virus (HBV) causes chronic hepatitis B, which can progress to liver cirrhosis and hepatocellular carcinoma. HBV has complex interactions with various cell organelles and proteins that ensure effective progeny virus production. We previously reported that a mitochondrial protein, TIMM29, should regulate the HBV life cycle through interactions with the HBV preS1 protein. Here, we established Halo-TIMM29wt-, Halo-TIMM29:∆99-192-, and Halo-TIMM29:92-194-expressing cells using TIMM29-knockout HB611 (TIMM29KO/HB611) cells, a stably HBV-producing cell line based on Huh6 cells. We found that HBV antigen expression and replication were downregulated in cells stably expressing full-length TIMM29, but not in those expressing TIMM29 deletion mutants. On the other hand, in the case of TIMM29-knockout C4 (TIMM29KO/C4), which is a human NTCP-expressing HepG2 cell line that is competent for HBV infection and amplification, these phenomena were not reproduced, except in full-length TIMM29 (Halo-TIMM29wt)-expressing cells. Using gene expression microarrays, we identified downregulation of ARRDC3 and BASP1 in TIMM29KO/HB611 and TIMM29KO/C4. It was suggested that TIMM29 localized at the mitochondrial inner membrane served as a signaling hub, orchestrating the activation of ARRDC3 and BASP1 expression to restrict HBV transcription. The expression of TIMM29 mutants in TIMM29KO/HB611 and TIMM29KO/C4 cells suggested that ARRDC3 was dependent on the HBV preS1-binding region of TIMM29 (amino acids 99-189). In contrast, BASP1 expression varied according to cell type, indicating additional regulatory mechanisms. Thus, this study should significantly advance our understanding of TIMM29-mediated inhibition of HBV amplification and lead to improvements in antiviral strategies and therapeutic interventions against HBV.