Transcutaneous immunization (TCI) is limited by poor permeation of macromolecules across the skin. Microneedle arrays form transient conduits and enhance the transport of vaccine molecules across the skin barrier without pain sensation. Here we investigated in mouse the immune responses after TCI using two model antigens, diphtheria toxoid (DT) and influenza subunit vaccine. The electric applicator enabled shorter microneedle (300 microm) to pierce mouse skin effectively, as shown by Trypan blue staining and trans-epidermal water loss measurement. The vaccines were topically applied with and without cholera toxin (CT) on microneedle-treated skin. In DT TCI, microneedle array pretreatment of the skin was essential to achieve substantial IgG and toxin-neutralizing antibody titers. Addition of CT further boosted the immune response to similar levels as observed after subcutaneous injection of AlPO4-adsorbed DT (DT-alum). In contrast, microneedle array pretreatment showed no effect on the immune response to plain influenza vaccine. This response was strongly improved by inclusion of CT, independent of microneedle treatment. These results indicate that TCI of DT and CT with microneedle treatment results in comparable protection as injection of DT-alum, and TCI of influenza vaccine adjuvanted with CT is superior to the injection of plain vaccine.