Insulin is a polypeptide drug and it is degraded by gastrointestinal enzymes, therefore, it cannot be used via oral route readily. There are only parenteral forms available in the market. The aim of this study was to investigate the effect of rectal and vaginal administration of various insulin gel formulations on the blood glucose level as alternative routes in rabbits. Chitosan gel (CH-gel) was used as a carrier; the penetration enhancing effect of sodium taurocholate and dimethyl-beta-cyclodextrin (DM-beta CD) was also investigated. CH-gel provided longer insulin release. The maximum decreasing effect on blood glucose level was observed with insulin-CH-gel containing 5% DM-beta CD. In conclusion, our results indicate that insulin may penetrate well through the rectal and vaginal mucosae from the CH-gel. DM-beta CD was also found to be a useful agent to enhance the penetration of insulin through rectal and vaginal membranes.