The aim of this article was to observe the intracellular insulin content of islets isolated from severely scalded and Exendin-4-treated rats and to evaluate the stimulation of insulin mRNA synthesis and secretion by β cells at different glucose concentrations and during different periods of time. A 50% TBSA full-thickness scalded rat model was used. Rats were treated with Exendin-4, followed by islet isolation and functional measurements. Serum was collected for detection of serum insulin, glucose, and glucagon levels. Intracellular insulin content was determined by transmission electron microscopy. Isolated islets were incubated with different glucose concentrations for 1 or 24 hours to assess the effect of scalding with or without Exendin-4 treatment on functional parameters. Insulin secretion was analyzed by enzyme-linked immunosorbent assay. Intracellular insulin and proinsulin content were analyzed by immunoprecipitation. Islet preproinsulin mRNA expression was examined by real-time polymerase chain reaction. Transmission electron microscopy analyses showed that severe thermal injury significantly reduced the number of insulin granules per micrometer2. Insulin secretion and intracellular insulin and proinsulin levels were markedly reduced in islets stimulated with different glucose concentrations; chronic high-glucose-stimulated islet preproinsulin mRNA expression was also impaired. Exendin-4 treatment after thermal trauma improved the number of intracellular insulin granules. Exendin-4 improved both insulin secretion and intracellular insulin reserves under different glucose stimulation conditions. Islet insulin mRNA expression was also restored by Exendin-4 treatment. Exendin-4 can restore the islet β cell insulin reserve following severe scald injury and may also improve insulin secretion, insulin reserve, and mRNA expression in islet β cells.