Hepatitis B virus (HBV) vaccination is regarded as the most economical and effective method for the prevention and control of HBV infection, a major global health problem. Previous studies have suggested that there may be sex‑specific differences regarding the immune response to the HBV vaccine in humans; however, the mechanisms associated with these sex‑specific differences are yet to be elucidated. In the present study, sex‑based immunological differences in mice following HBV vaccination were investigated to determine the mechanisms underlying sexual dimorphism, with the aim of identifying potential targets for clinical intervention. Balb/c mice (n=6) were vaccinated intramuscularly on 3 different days (days 0, 14 and 28) with the HBV vaccine. Sera were analyzed via ELISA for the presence of HBV surface antigen (HBsAg)‑specific immunoglobulin G (IgG), and of different IgG subtypes, 3 weeks following the third injection. Enzyme‑linked immunosorbent spot assays were conducted to determine interleukin‑4/interferon‑γ secretion. Immunological memory stimulated by the vaccine was detected via flow cytometry analysis and ELISA 1 week following the booster immunization. The seroconversion of the treated female group was higher compared with the male group at one week following the second vaccination. Female mice exhibited significantly increased HBsAg antibody titers compared with males at 1‑5 weeks following the third vaccination. Sera obtained from vaccinated female mice exhibited markedly increased titers of IgG1 and IgG2b compared with those from male mice. Furthermore, female mice exhibited elevated cytotoxic T lymphocyte responses and immune memory. Collectively, the results of the present study indicated that sex‑based immunological differences affected the dynamics and characteristics of the immune response in mice immunized with the HBV vaccine.