In this study, we aimed to develop a novel protein - nanoencapsulated system for oral administration. For this purpose, insulin was selected as the model drug. Insulin loaded chitosan nanoparticles (INS-CS-NPs) were obtained by ionic gelation between chitosan (CS) and sodium tripolyphosphate (TPP). Afterwards, as a novel strategy the nanoparticles were loaded into the inner phase of prepared water in oil micro emulsion to provide sustained released, increased in vivo stability and enhanced drug absorption in the gastrointestinal tract. By this way, INS-CS-NPs encapsulated in microemulsion (INS-CS-NP-ME) was formed. The in vitro release properties of formulations with different INS:CS and CS:TPP ratios were investigated. In vitro release study in pH 2.5 revealed that insulin release was significantly low under higher CS ratios (p < 0.05). Circular dichroism analyses showed that the conformational stability of insulin was not affected from preparation process. Furthermore, in vivo experiments in Wistar Albino rat model demonstrate that INS-CS-NP-ME effectively reduced blood glucose levels over a period of 8 h after oral administration. Based on these findings, we propose that the developed INS-CS-NP-ME system can be a promising alternative dosage form for oral protein delivery. (C) 2016 Elsevier B.V. All rights reserved.