Exendin-4 (ex-4) is a peptide molecule that regulates blood glucose levels without causing hypoglycemia by providing insulin secretion from beta cells in the pancreas. Self-nanoemulsifying drug delivery systems (SNEDDS) attract attention for oral administration of therapeutic peptide/proteins because they protect therapeutic peptide/proteins from the gastric environment, reduce changes due to food effects, are easy to prepare and scale-up. Ex-4 has no commercial form that can be administered orally. In this study, the cytotoxicity, cellular uptake, and insulin secretion of ex-4 and ex-4/chymostatin (chym) SNEDDS were investigated on INS-1E rat pancreatic beta cells. The effect of ex-4 and ex-4/chym SNEDDS on cell viability in INS-1E cells increased when the dilution ratio higher. Ex-4 and ex-4/chym SNEDDS increased insulin levels in 2.8 mM (low-dose) glucose-induced INS-1E cells 2.21-fold and 2.17-fold compared to control, respectively. Ex-4 and ex-4/chym SNEDDS increased insulin levels in 16.7 mM (high dose) glucose-induced INS-1E cells compared to control, respectively. In cellular uptake studies, coumarin-6 solution penetrated the apical membrane of INS-1E cells and remained in the cytoplasm, while coumarin-6 loaded SNEDDS were visualized in the nuclei of the cell. These findings will likely be useful in the development of new formulations for the oral administration of peptides/proteins.