Background and Aims: Chronic hepatitis B virus (HBV) in-fection is a global public health challenge. HBV reactivation usually occurs in cancer patients after receiving cytotoxic chemotherapy or immunosuppressive therapies. Romidepsin (FK228) and vorinostat (SAHA) are histone deacetylase in-hibitors (HDACi) approved by the Food and Drug Administra-tion as novel antitumor agents. The aim of this study was to explore the effects and mechanisms of HDACi treatment on HBV replication. Methods: To assess these effects, hu-man hepatoma cell lines were cultured and cell viability after FK228 or SAHA treatment was measured by the CCK-8 cell counting kit-8 assay. Then, HBV DNA and RNA were quantified by real-time PCR and Southern blotting. Furthermore, analy-sis by western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and flow cytometry was per-formed. Results: FK228/SAHA treatment significantly pro-moted HBV replication and biosynthesis in both HBV-replicat-ing cells and HBV-transgenic mouse model. Flow cytometry assay indicated that FK228/SAHA enhanced HBV replication by inducing cell cycle arrest through modulating the expres-sion of cell cycle regulatory proteins. In addition, simultane-ous inhibition of HDAC1/2 by FK228 promoted HBV replica-tion more effectively than the broad spectrum HDAC inhibitor SAHA. Conclusions: Overall, our results demonstrate that cell cycle blockage plays an important role in FK228/SAHA-enhanced HBV replication, thus providing a potential avenue for rational use of HDACi in patients with chronic hepatitis B.