Objective To evaluate the influence of hepatitis B virus (HBV) genome nucleotide A1846T mutation on the viral replication capacity and the transcription activity of HBV core promoter (CP) in vitro. Methods A total of 385 patients with hepatitis B admitted to the 302 Hospital of PLA were enrolled in the study, including 116 with moderate chronic hepatitis B (CHB-M), 123 with severe chronic hepatitis B (CHB-S), and 146 with acute-on-chronic liver failure (ACLF). Serum HBV DNA was isolated and full-length HBV genome was amplified. The incidence of A1846T was analyzed. Full-length HBV genomes containing 1846T mutation were cloned into pGEM-T easy vector, and the counterpart wild-type 1846A plasmids were obtained by site-directed mutagenesis. The full-length HBV genome was released from recombinant plasmid by BspQ Ⅰ/Sca Ⅰ digestion, and then transfected into HepG2 cells. Secreted HBsAg level and intracellular HBV core particles were measured 72 hours post-transfection to analyze the replication capacity (a 1.0-fold HBV genome model). 1846 mutant and wild-type full-length HBV genomes were extracted to amplify the fragment of HBV CP region, and the dual luciferase reporter of the pGL3-CP was constructed. The luciferase activity was detected 48 hours post-transfection. Results The incidence of A1846T mutation gradually increased with the severity of hepatitis B, reaching 31.03%, 42.27%, and 55.48% in CHB-M, CHB-S and ACLF patients respectively (P＜0.01). The replication capacity of 1846T mutants, level of secreted HBsAg, and transcriptional activity of CP promoter were increased by 320%, 28% and 85% respectively, compared with 1846A wild-type strains. While the more common double mutation A1762T/G1764A in CP region was increased by 67%, 9% and 72% respectively, compared with its counterpart wild-type strains. A1846T had a greater influence on viral replication capacity in vitro. Conclusions A1846T mutation could significantly increase the replication capacity of hepatitis B virus, secretion of HBsAg and transcription activity of CP promoter, and cis-activate the downstream gene transcription. The finding indicates that HBV genome A1846T mutation might play a role in liver disease progression.