To investigate the effects of anti-CD20 monoclonal antibody (mAb) combined with IL-10 mediated by adenovirus on the regeneration of pancreatic β cells in nonobese diabetic (NOD) mice. Serum IL-10, TGF-β, TNF-α, IFN-γ, C-peptide, and insulin were determined using commercial ELISA. Western blot assay and real-time PCR were performed to determine the expression of mRNA and protein expression, respectively. Besides, immunohistochemistry and immunofluorescence were performed. Immunohistochemisty assay indicated Ngn3 expression showed decrease in the anti-CD20+IL-10 group compared with the other groups (p < 0.05). Immunofluorescence assay showed Pdx-1 and insulin coexpressing cells showed increase in the anti-CD20+IL-10 group. Real-time PCR indicated that the expression of Pdx-1, Pax4, Nkx6.1, and Mafa mRNA showed significant increase in the anti-CD20+IL-10 group compared with the other groups (p < 0.05). The expression of Pdx-1, Pax4, Nkx6.1, and Mafa protein was significantly upregulated in the anti-CD20+IL-10 group compared with that of the other groups (p < 0.05). Anti-CD20 combined with IL-10 promoted insulin secretion in NOD mice and the controlling of blood glucose. Besides, the combination induced the decrease of TNF-α and IFN-γ, and the elevation of TGF-β. Besides, it may activate the Pdx-1-Ngn3-Pax4-Nkx2.2-Nkx6.1 signaling pathway, and trigger the regeneration of pancreatic β cells.