Objective This study was conducted to establish a stable and highly efficient method for isolation and purification of islets in NOD mice and to evaluate in vitro and in vivo characterization. Methods The pancreatic islets were isolated from the pancreas by modified collagenase digestion and Ficoll density gradient centrifugation method. The endocrine secretory function was assessed by insulin secretion in either low or high glucose stimulation. To evaluate the functionality of the graft, body weight and blood glucose were monitored, and IVGTT was performed. In addition, to assess survival of the implanted islets, HE staining and insulin immunostaining of the graft were performed. Results the average islet yield was (116 ? 12) islets/pancreas and purity was greater than 90%. The islets isolated from NOD mice are poorly responsive to glucose challenge, compared with islets from Kunming mice. Blood glucose levels and body weight change of islet-transplanted diabetic mice improved significantly compared with the sham-operated mice. In addition, blood glucose levels in vivo after an IVGTT also significantly improved. However, these improvements could only be maintained for 2 weeks. Furthermore, HE staining and immunostaining assay demonstrated that there were insulin-positive cell clusters and infiltration of lymphocytes in the graft bearing kidney. Conclusions a large number of quality islets can be isolated and purified from NOD mice by using the modified mouse islet isolation method, which can be develop therapeutic strategies to protect transplanted islets from rejection and autoimmune attack.