Objective: Several studies demonstrated some alterations in immune system of patients with β-thalassemia major. The aim of study was to investigate the function and number of lymphocyte subsets of patients with β-thalassemia major.Materials and Methods: Quantitative and qualitative assessment of cellular and humoral immune system and population of natural killer lymphocytes in patients with β-thalassemia major and similar age and sex matched healthy controls were done. Complete blood count and serum iron and ferritin levels were also measured. Data was analyzed by SPSS (V.16) software. Results: We did not find any alteration in number and function of B and T lymphocytes. However, natural killer cells level in thalassemia group was significantly lower than control group (6.54±2.87% vs. 9.13±4.01%; P=0.006). Number of T helper cells in thalassemia patients with spelenectomy was significantly lower than patients without splenectomy (31.8±6.55% vs. 40.3±9.2%; P=0.02). Conclusion: In our study the patients with β-thalassemia major had lower natural killer (NK) lymphocytes than healthy control.