Objective：This study aims to explore the characteristics of follicular cytotoxic T cells（TFC）and the role they played in the natural course of autoimmune diabetes in non-obese diabetic（NOD） mice. Methods：The spleen，lymph nodes and thymus lymphocytes of NOD mice in 4 weeks，10 weeks，15 weeks，20 weeks，25 weeks，and hyperglycemia（Hi） groups were separated and stained by flow cytometry antibodies. Through flow cytometry，we observed and compared the distribution of TFC and CXCR5-CD8+T cells in different immune organs，the changes of cell proportion in the natural course of autoimmune diabetes，the expression levels of degranulation markers cluster of differentiation 107a（CD107a） and recombinant human killer cell lectin-like receptor subfamily G，member 1（KLRG1），inhibitory molecules programmed cell death protein 1（PD-1） and T cell immunoglobulin mucin 3（TIM-3），and the ability of releasing interferon γ（IFN-γ）and granzyme B（GzmB）. Results：The results of this study illustrated that the expression and distribution of TFC was different in various immune organs of NOD mice. The expression levels in the spleen and lymph nodes were equivalent，but there was almost no expression in the thymus. Before the blood sugar of NOD mice increased，the proportion of TFC cells showed a gradual upward trend along with the increasing age，which decreased after the onset. Compared with CXCR5-CD8+T cells，the proportion of TFC was significantly lower，but the inhibitory molecules PD-1，TIM-3 and the degranulation marker CD107a expressed on TFC were significantly higher. At the same time，TFC has a stronger ability to release GzmB［TFC（3.56±2.1）%；CXCR5-CD8+T cell（0.78±0.39）%，P < 0.05］and IFN-γ［TFC（43.65±12.25）%；CXCR5-CD8+T cell（21.92±3.72）%，P < 0.05］than CXCR5-CD8+ T cells. Conclusion：TFC exists and participates in the natural course of autoimmune diabetes in NOD mice，and promotes immune damage in NOD mice through cytotoxic effect.