The aim of this report was to develop a dissolving microneedle (DM) application system, where 225-300 insulin-loaded DMs were formed on a chip. After the heat-sealed sheet is removed, the system covered with the press-through package layer is put on the skin. By pressing with the hand, insulin DMs were inserted into the skin.Factors affecting the penetration depth of DM were studied using applicator in vitro and in vivo experiments. The penetration depth was determined for rat and human skin. Two-layered DM array chips were prepared to obtain complete absorption of insulin and administered to the rat abdominal skin. Plasma glucose levels were measured for 6 h. By comparing the hypoglycemic effect with that obtained after subcutaneous injection, relative pharmacological availability (RPA) was determined.The penetration depth increased from 21 ± 3 μm to 63 ± 2 μm in proportion to application speed to isolated rat skin, at 0.8-2.2 m/s. Human skin showed similar results in the penetration depth. The in vivo penetration depth was dependent on the force (0.5-2.5 N) and duration (1-10 min), as the secondary application force. The penetration depth was 211 ± 3 μm with a duration of 3 min in the in vivo rat experiment. DM array chips having an insulin-loaded space of 181.2 ± 4.2 and 209 ± 3.9 μm were evaluated in the rat. RPA values of insulin from DMs were 98.1 ± 0.8% and 98.1 ± 3.1%, respectively.These results suggest the usefulness of the two-layered DM application system for the transdermal delivery of insulin.