Polyclonal T regulatory cells (Treg - CD4CD25CD127Foxp3) are used in several protocols for the treatment of type 1 diabetes (T1D)， multiple sclerosis and graft-versus host disease in clinical trials. However， general opinion is that autoantigen-specific Treg could be more efficient in autoimmunity suppression due to their direct effect on pathogenic autoantigen-specific effector T cells. This study describes isolation and expansion of insulin-specific Treg in vitro. Insulin-specific Treg are uniformly distributed in lymphoid tissues however their number is extremely low. To enrich the proportion of insulin-specific Treg， pure CD4 cells were co-cultured with insulin B chain peptide-loaded dendritic cells， isolated from mice that develop T1D spontaneously - NOD mice. Insulin-specific CD4 cell expansion peaked after 48 h of incubation and was in favour of Treg. These cells were then sorted using insulin peptide-loaded MHC class II tetramers and cultured in vitro for 48 h in the presence of TCR stimulators， TGF-β and IL-2. The proportion of gained insulin-specific cells with T regulatory phenotype (CD4CD25CD127GITRFoxP3) was in average between 93% and 97%. These cells have shown potent in vitro suppressive effect on T effector cells， produced IL-10 and TGF-β and expressed PD-1 and CD39. Further proliferation of these insulin-specific Treg required the presence of dendritic cells， anti-CD3 antibody and IL-2. This study provides new， reproducible experimental design for the enrichment and expansion of insulin-specific Treg that can be used for the cell-based therapy of autoimmunity.