Regulatory T cells (Tregs) have key roles in the immune response by suppressing the differentiation and proliferation of various immune cells. The beneficial effects of docosahexaenoic acid (DHA) have been described for many diseases; however, the mechanism by which it modulates the immune system is poorly understood. Therefore, the aim of this study was to examine whether DHA suppresses allergic reactions and upregulates the generation of CD4+Foxp3+ T cells. We also examined the effects of transfusing interleukin-10/transforming growth factor-β (TGF-β)-modified macrophages (M2 macrophages) treated with DHA into a mouse model of atopic dermatitis. Here, we show that administration of DHA upregulates the generation of TGF-β-dependent CD4+ forkhead box protein 3 (Foxp3+) Tregs. DHA induced T-cell hypo-responsiveness and downregulated cytokines associated with T helper (Th)-1, Th2, and Th17 cells. The differentiation of Foxp3+ Tregs into CD4+ T cells was directly mediated by DHA-M2 macrophages, which deactivated effector macrophages and inhibited CD4+ T-cell proliferation. DHA showed therapeutic effects in mice with experimental atopic dermatitis. These results show that DHA enhances the function of M2 macrophages and that the generation of Tregs effectively protects mice against an inflammatory immune disorder. Thus, DHA may be a useful therapeutic strategy for treating chronic inflammatory diseases.