Objective This study was conducted to establish a stable and highly efficient method for isolation and purification of pancreatic islets from NOD mice and to evaluate their characteristics in vitro and in vivo. Methods The islets were isolated from mouse pancreas using modified collagenase digestion and Ficoll density gradient centrifugation. The endocrine secretory function was assessed by insulin secretion in either low or high dose glucose stimulation. To evaluate the function of the graft, body weight and blood glucose were monitored, and IVGTT was performed. In addition, to assess survival of the implanted islets, Pathology using HE staining and insulin immunostaining of the graft were performed. Results The average islet yield was 116 ±12 islets/ pancreas and purity was higher than 90%. Compared with islets from Kunming mice, the islets isolated from NOD mice were poorly responsive to glucose challenge. Blood glucose levels and body weight changes of the islet-transplanted diabetic mice were significantly improved compared with the sham-operated mice. In addition, blood glucose levels in vivo after an IVGTT also significantly improved. However, these improvements were only main-tained for 2 weeks. Furthermore, HE staining and immunostaining assays demonstrated that there were insulin-positive cell clusters and lymphocyte infiltration 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 used to develop therapeutic strategies to protect transplanted islets from rejection and autoimmune attack.