Abstract: Objective To isolate human insulinoma cells, and investigate its functional characteristics. Methods Primary human insulinoma cells were isolated by using collagenase type P, and then purified by repeatedly selective cell culture. The cellular autofluorescence was checked by fluorescent microscope. Expression of insulin and islet cell autoantigens such as IA-2, IAR and ICA were checked by RT-PCR and Western blot. The cytotoxicity induced by anti-human IA-2 antibody was analyzed by MTT assay. Results The human insulinoma cells could form islet-like structure under the condition of suspension culture and presented autofluorescence under fluorescent microscope. The human insulinoma cells expressed lower level of insulin mRNA and protein as compared with a rat insulinoma cell line, INS-1. The insulinoma cells were also expressed autoantigens of islet cells such as IA-2, IAR and ICAp69. A dose dependent cytotoxicity was found when the insulinoma cells incubated with anti IA-2 antibody. Conclusion Human beta cells derived from insulinoma could be purified by selective cell culture. Since the insulinoma cells expressed insulin and islet cell autoantigens, it could be used as cell model for investigating the related mechanisms of type I diabetes.