Transplantation of pancreatic islets is a promising therapeutic treatment for type 1 diabetes mellitus. For clinical and experimental transplantation, a large number of pure pancreatic islets are required for transplantation. Thus, the improvement of islet isolation and purification techniques are crucial. In this context, iodixanol-based solution, successfully used for the purification of porcine islets, seems to be a possible alternative to Ficoll for purification of islets. The aim of this study was to test the efficacy of iodixanol compared with Ficoll density gradients for the purification of rat pancreatic islets. Twelve Wistar rats were used for isolation and purification of pancreatic islets. Pancreata were digested with Liberase R1 and islets purified by two gradients: Ficoll or iodixanol gradient. The number and the purity of the pancreatic islets were assessed. To analyze the response of isolated pancreatic islet to glucose challenge, in vitro experiments were performed by measuring the insulin concentration in the Supernatant. The results demonstrated that the iodixanol gradient provided a higher purity of pancreatic islets compared to the Ficoll gradient. In addition, the rat islet yield by iodixanol gradient was significantly higher compared to a Ficoll gradient (751 +/- 16 versus 464 +/- 19 pancreatic islets, respectively; P < .001). The viability of pancreatic islets isolated by an iodixanol gradient was confirmed by high glucose challenge, with more than twofold higher increase in insulin secretion. The present study demonstrated that iodixanol density gradient overcomes Ficoll density gradient, providing a greater number of pure and functional rat pancreatic islets.