Objective：To optimize the isolation and purification of rat islets. Methods：Pancreas of SD rats were digested by collagenase V and purified by Histopaque-1077. The islets were stained with trypan blue to identify the viability, and stained with dithezone to count the yield. Glucose stimulated insulin secretion（GSIS） of islets was performed in vitro. Allogeneic islets transplantation via portal vein was performed in STZ-induced diabetic SD rats and the blood glucose level was observed. Results：The average yield was 646 ± 67 perrat and the livability rate was above 90%. GSIS showed that SI（stimulating index） was 2.6, 1.7 and 1.4 at the 1 st, 3 rd, 5 th respectively. There was significant difference between low glucose groups and high glucose groups（P < 0.05）. After transplantation, the blood glucose decreased to 11.1 mmol／L at the 2nd day and kept below 15 mmol／L for 8 days. Conclusion：Collagenase V digestion combined with Histopaque-1077 purification is an effective method for rat islets isolation, and the islet yield is high with good function.