All the immune cells such as T cells, macrophages and dendritic cells (DCs), have ACh-synthesizing ability and express α7 nAChRs involved in regulation of proliferation, and synthesis of antigen-specific antibodies and proinflammatory cytokines. We investigated role of α7 nAChRs on APCs in regulation of CD4⁺ T cell differentiation. Spleen cells, including naïve CD4⁺ T cells and APCs (macrophages and DCs), were isolated from ovalbumin (OVA)-specific TCR transgenic DO11.10 mice, and cultured with OVA in the presence of GTS-21, an α7 nAChR agonist. GTS-21 suppressed the OVA-activated CD4⁺ T cell differentiation into regulatory T cells (Tregs), Th1, Th2 and Th17 cells. GTS-21 inhibited the production of Th cytokines (IFN-γ, IL-4 and IL-17). GTS‑21 inhibited differentiation into Tregs of OVA-induced CD4⁺ T cells co-cultured with APCs from the wild-type (WT) mice but did not affect differentiation of T cells co-cultured with APCs from α7 nAChR-deficient mice. These results suggest a critical role of α7 nAChRs on APCs in regulation of CD4⁺ T cell differentiation, and that α7 nAChR agonists and antagonists could be potentially useful agents for immune response modulation and enhancement.