Atrazine (ATR), one of the most widely used herbicides worldwide, has caused a series of toxicological and environmental problems. This study sought to investigate the effects of ATR on the immune system of mice. Four-week-old female C57B l/6 mice were treated with 5, 25, and 125 mg/kg ATR for 28 days. On day 29, blood was collected and the spleen was harvested to detect lymphocyte transformation, natural killer (NK) cell activity, cellular phenotypes, and cytokines. Results indicated that the thymus and spleen weights decreased after ATR treatment, and the spleen was found to be more sensitive to ATR than the thymus. Decreases in lymphocyte transformation and NK cell activity were also observed in mice treated with 25 mg/kg ATR and 125 mg/kg ATR compared to the control group. In addition, there were also alterations of lymphocyte phenotypes in the spleen, and the percentages of CD3+ and CD4+ cells decreased in mice treated with 25 mg/kg ATR and 125 mg/kg ATR compared to the control group. Moreover, serum interleukin-4 level decreased significantly after treatment with 25 mg/kg and 125 mg/kg ATR, but ATR did not affect the expression of interleukin-2, interferon-γ, and tumor necrosis factor-α. These results suggest that ATR may have induced damage in spleen cells. As ATR is an environmental contaminant, its immunosuppressive action raises concerns that it may potentiate clinical conditions such as tumors, inflammation, and infections. Thus, it needs to be carefully monitored and studied.