Aims: Sirtuin 2 (Sirt2), an NAD+-dependent protein deacetylase, deacetylates tubulin, AKT, and other proteins. Previously, we showed that Sirt2 isoform 1 (Sirt2.1) increased replication of hepatitis B virus (HBV). Methods: Here, we show that HBV replication upregulates expression of Sirt2 primary and alternatively spliced transcripts, and their respective isoforms 1, 2, and 5. Since Sirt2 isoform 5 (Sirt2.5) is a catalytically inactive nuclear protein with a splicedout nuclear export signal (NES), we speculated that its different localization may affect its activity. Results: Overexpression of Sirt2.5 reduced expression of HBV mRNAs, replicative intermediate DNAs, and covalently closed circular DNA (cccDNA), an activity opposite to that of Sirt2.1 and Sirt2.2. Unlike the Sirt2.1-AKT interaction, the Sirt2.5-AKT interaction was weakened by HBV replication. Unlike Sirt2.1, Sirt2.5 activated the AKT/GSK-3ß/ß-catenin signaling pathway very weakly and independently of HBV replication. When the NES and an N-terminal truncated catalytic domain were added to the Sirt2.5 construct, it localized in the cytoplasm and increased HBV replication (like Sirt2.1 and Sirt2.2). Chromatin immunoprecipitation assays revealed that more Sirt2.5 was recruited to cccDNA than Sirt2.1. Also, recruitment of histone lysine methyltransferases (HKMTs) such as SETDB1 and SUV39H1, EZH2, and PR-Set7, and their respective transcriptional repressive markers H3K9me3, H3K27me3, and H4K20me1, to cccDNA increased in Sirt2.5-overexpressing cells. Among these, the Sirt2.5-PR-Set7 and -SETDB1 interactions increased upon HBV replication. Conclusions: These results demonstrate that Sirt2.5 reduces cccDNA levels and viral transcription through epigenetic modification of cccDNA via direct and/or indirect association with HKMTs, thereby exhibiting anti-HBV activity.