Immune escape mutants with mutations in the hepatitis B surface antigen (HBsAg) major hydrophilic region (MHR) often emerge in association with diagnostic failure or breakthrough of HBV infection in patients with anti-HBs antibodies. Some mutants harboring substitutions to Asn in HBsAg MHR may have an additional potential N-glycosylation site. We have previously showed that sT123N substitution could generate additional N-glycosylated forms of HBsAg. In the present study, 1.3-fold-overlength HBV genomes containing the sT123N substitution were digested from the pHBV1.3-sT123N construct and subcloned into the pAAV vector to generate pAAV1.3-sT123N for hydrodynamic injection (HI) in mice. Viral expression and replication were phenotypically characterized by transient transfection. The results demonstrated that sT123N substitution impaired virion secretion, resulting in intracellular retention of HBcAg. Using the HBV HI mouse model, we found that mice mounted significantly stronger antibody responses to HBsAg and HBcAg, which accelerated HBsAg clearance. Thus, additional N-glycosylation generated by amino acid substitutions in HBsAg MHR may significantly modulate specific host immune responses and influence HBV infection in vivo. Our results help further the understanding of the role of immune escape mutants with N-linked glycosylation in the biology of HBV infection.