To develop an effective, convenient and stable mucosal vaccine against hepatitis B virus (HBV), the mannose-PEG-cholesterol/lipid A-liposomes (MLLs) loaded with HBsAg were prepared by the procedure of emulsification-lyophilization and, subsequently, filled into the microholes of microneedle array reverse molds and dried to form the proHBsAg-MLLs microneedle arrays (proHMAs).The proHMAs were stable even at 40 ℃ for up to 3 days and hard enough to pierce porcine skin but, upon rehydration, rapidly dissolved recovering the HBsAg-MLLs without obvious changes in size and antigen association efficiency.Notably, immunization of mice only once with the proHMAs at oral mucosa induced robust systemic and widespread mucosal immunoresponses, as evidenced by the high levels of HBsAg-specific IgG in the sera and IgA in the salivary, intestinal and vaginal secretions.In addition, a strong cellular immunity against HBV had been established through a mixed Thl/Th2 response, as confirmed by a significant increase in CD8+ T cells as well as the enhanced levels of IgG2a and IFN-γin the treated mice.Thus, the proHMAs can be conveniently vaccinated via oral mucosal route to set up a multiple immune defense against HBV invasion and, in addition,may be a stable HBV vaccine applicable in the controlled temperature chain for wide distribution.