Hepatitis B virus (HBV) biosynthesis is primarily restricted to hepatocytes due to the governing of liver-enriched nuclear receptors (NRs) on viral RNA synthesis. The liver-enriched NR hepatocyte nuclear factor 4α (HNF4α), the key regulator of genes implicated in hepatic glucose metabolism, is also a primary determinant of HBV pregenomic RNA synthesis and HBV replication. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) coactivates and further enhances the effect of HNF4α on HBV biosynthesis. Here, we showed that the acetyltransferase General Control Non-repressed Protein 5 (GCN5) acetylated PGC1α, leading to alteration of PGC1α from a transcriptionally active state into an inactive state. As a result, the coactivation activity of PGC1α on HBV transcription and replication was suppressed. Apparently, an acetylation site mutant of PGC1α (PGC1αR13) still had coactivation activity as GCN5 could not suppress the coactivation activity of the mutant. Moreover, a catalytically inactive acetyltransferase mutant GCN5m, due to the loss of acetylation activity, failed to inhibit the coactivation function of PGC1α in HBV biosynthesis. Our results demonstrate that GCN5, through its acetyltransferase activity, inhibits PGC1α-induced enhancement of HBV transcription and replication both in vitro and in vivo.