Abstract Exendin-4 is a 39-amino acid peptide that shares several glucoregulatory activities with the mammalian incretin hormone, glucagon-like peptide-1 (GLP-1). The synthetic form of exendin-4, exenatide, was approved as adjunctive therapy by subcutaneous injection for patients with type 2 diabetes failing to achieve glycemic control with oral antidiabetic agents. Basically, the oral route is the most convenient way of drug administration for patients because it can avoid pain and reduce the complex complication. Base on our previous study, we developed nanoparticles (NPs) composed of chitosan (CS), poly-γ-glutamic acid (γPGA) with Fe ions as the carrier for exendin-4 delivery via the oral route. The size and zeta potential of the prepared NPs were ~300nm and ~30mV, respectively. The exendin-4 loaded in NPs could get ~60% loading efficiency (LE) and ~15% Loading Content (LC). To protect NPs in the acidic stomach environment, gelatin hard capsules filled with NPs were coated with enteric coating polymer. The enteric coating polymer, Eudragit L55-100, was dissolved beyond pH 6.0 that mimetic the pH environment of duodenum in the GI track. In the dissolution study, the test capsule could be dissolved immediately as it reached the duodenal region and NPs could then release exendin-4 into the systemic circulation via the opened paracellular pathway. For the in vivo study, the biodistribution of exendin-4 loaded NPs following the oral administration to rats was performed using the single-photon emission computed tomography (SPECT)/computed tomography (CT). The results obtained in the SPECT/CT study indicate that the orally administered exendin-4 was significantly absorbed into the systemic circulation. There was very high circulating exendin-4 (~48%) in the peripheral tissue and plasma (PP) at 2 h post ingestion. In the pharmacodynamic (PD) and pharmacokinetic (PK) evaluation in a mild diabetic rat model, the orally administered exendin-4 loaded NPs produced a slower hypoglycemic response for a prolonged period of time. The relative bioavailability of exendin-4 orally delivered by test NPs in gelatin capsules coated with 10% Eudragit was found to be 13.7 ± 2.1%. The results suggest the suitability of the NP system to be used as a non-invasive alternative for the basal exendin-4 therapy.