Introduction Allergic conjunctivitis (AC) is one of the most frequent ophthalmological diseases. AC is characterized by ocular surface inflammation and systemic immune dysregulation with low frequency of CD4+CD25+FOXP3+ regulatory T cells and higher frequency of circulating effector T cells. It has been reported that α-melanocyte stimulating hormone (α-MSH) constitutes one of the principal mechanisms of immune regulation in the anterior chamber of the eye and the ocular surface, promoting the differentiation of Tregs in a murine model of experimental autoimmune uveitis and suppressing autoreactive T cells in a murine model of experimental autoimmune encephalomyelitis. This work aimed to evaluate the involvement of α-MSH in Tregs induction in cells obtained from patients with AC. Methods Peripheral blood mononuclear cells (PBMC) were obtained from blood sampling after assent consent was obtained from pediatric patients sensitized to house dust mite (HDM) and diagnostic with AC. PBMC were cultured with HDM and/or α-MSH. After 72h of culture, PBMC were harvested and stained with anti-CD4-AmCyan, anti-CD25-PE, anti-FOXP3-FITC and anti-CD69-APC-Cy7 and analyzed by flow cytometry. IL-2, IL-4, IL-6, IL-10, IL-17, TNF-a and IFN-γ were determined by cytometric bead arrays. Results A significant increase in the frequency of CD4+CD25+FOXP3+ T cells and a significant decrease in the frequency of CD4+CD69+ cells were observed in HDM+α-MSH stimulated cells, when compared with HDM. Remarkably, IL-4 showed a significant decrease in cultured cells stimulated with HDM+α-MSH. Conclusion These results suggest that a-MSH inhibit the IL-4 production, reestablishing the development of Treg cells and control pathogenic CD4+CD69+ T cells.