To explore the feasibility of the application of (99m)Tc-DTPA-Nateglinide as a nuclear medicine imaging agent for evaluating pancreatic B cell function. (1) Distribution of the experiment: Forty-two mice were selected and divided into seven groups. Each mice was injected with 3.7 MBq (100 μCi) of (99m)Tc-DTPA-NGN2 from the vena caudalis and was sacrificed by bloodletting at five minutes, 15 minutes, 30 minutes, one hour, two hours, four hours and six hours, respectively. Then, their tissues and organs such as the heart, liver, spleen, brain, kidneys, bones, small bowels, stomach and pancreas,and blood were collected, weighted, and their radioactivity was tested. Subsequently, the percentage injection dose rate (%ID/g) per gram of tissue was calculated. (2) Imaging experiment: Thirty-five mice were selected and divided into seven groups. Each was injected with 18.5 MBq (100 μCi) of (99m)Tc-DTPA-NGN2 from the vena caudalis and imaging were conducted at the same time as above. (3) Forty-eight Wistar rats were attained and randomly divided into four groups. The first group served as the healthy control group, while the second, third and fourth groups were diabetic model groups induced by intraperitoneally injecting STZ at different doses. Each group was injected with (99m)Tc-DTPA-Nateglinide from the vena caudalis, and radiological evaluations were conducted at 30 minutes, one hour, 1.5 hours and two hours, respectively. The data obtained were estimated using a correlation comparison with the levels of insulin and immunohistochemical count of beta cells. The (99m)Tc-DTPA-Nateglinide demonstrated good imaging in the pancreases of mice and rats, and was positively correlated to the level of insulin and the number of pancreatic beta cells. Pancreatic beta cell imaging using (99m)Tc-DTPA-Nateglinide may be a method to evaluate pancreatic beta cell function.