Background: Drugs used in cancer treatment specifically kill T regulatory cells. Objective: To determine different phenotypes of T regulatory cells during the maintenance phase chemotherapy for pediatric acute lymphoblastic leukemia (ALL). Materials: We evaluated the percentages of regulatory T cells by flow cytometry. Soluble CTLA-4 (sCTLA-4) in plasma was evaluated by ELISA assay. Results: Increased percentages of CD4+CD25+ T cells, CD4+CD39+ T cells, CD4+Foxp3+ T cells, and CD4+CD25High T cells were observed in children with ALL in comparison to healthy controls. In addition, the ALL patients with >12 months of therapy showed increased CD4+CD39+ T cells compared to the ALL patients with ≤12 months and healthy controls. Similarly, the CD4+CD25+ T cells and CD4+Foxp3+ T cells increased according to maintenance therapy time. Conclusion: Our results showed increased percentages of regulatory T cells in pediatric ALL patients despite chemotherapy, which might be compromising the anti-leukemic cellular immune response.