Type 1 diabetes (T1D) is an autoimmune disease characterized by a selective destruction of insulin-secreting β-cells. Both T cells and B cells serve a crucial role in pathogenesis and development of T1D. CD20 is a specific membrane antigen of B lymphocytes, while interleukin (IL)‑10 is an important cytokine secreted by T helper 2 cells and has a short half‑life in vivo. The combined effect of anti‑CD20 and IL‑10 on immune function of mice with T1D remains unknown. In the present study, 30 non‑obese diabetic (NOD) mice were treated with anti‑CD20 and adenoviral vector‑mediated interleukin‑10 (Ad‑mIL‑10) therapy. Alterations in CD4+, CD8+, CD4+CD25+Foxp3+ T cells, T‑box expressed in T‑cells (T‑bet), GATA‑binding protein‑3 (GATA‑3) interferon‑γ (IFN‑γ) and IL‑4 were detected by flow cytometry, reverse transcription‑quantitative polymerase chain reaction in NOD mice spleen tissue. The present results suggested that anti‑CD20 and IL‑10 treatment in NOD mice can modulate the immune functions by upregulating GATA‑3 and IL‑4 expression as well as downregulating T‑bet and IFN‑γ expression, which are involved in the pathogenesis of T1D. The current findings may provide a potential method for T1D treatment and a novel preventive therapy for T1D. Combination of anti‑CD20 and Ad‑mIL‑10 treatment had not only immune regulatory effects but also protective effects on islet β‑cells in NOD mice with T1DM at the early stages, by regulating T‑bet/GATA‑3 expression and Th1/Th2 cell differentiation, which has the potential for diabetes prevention and therapy.