It is well known that ozone (O3), a potent reactive oxidant and air pollutant, induces respiratory inflammation and hyperresponsiveness upon inhalation. It was previously shown that O3 exposure (0.6 ppm, 10 h/day for 15 days) not only results in local bronchial inflammation, but also affects the nervous system and thymocyte proliferation, and places mice under oxidative stress. In the present study, data showed that O3 exposure could impair both the natural killer (NK) cell activity and the proliferation potential of spleen T cells to a specific antigen stimulus. Immunological function assays indicated that O3 exposure attenuated the proliferation of spleen mononuclear cells induced by concanavalin A and decreased CD4+ and CD28+ lymphocyte subsets. However, supplementation with natural antioxidants protected mice from O3-induced dysfunction of splenocyte proliferation. Meanwhile, O3 exposure resulted in a decline of mitogen-induced IL-2 production in splenocytes. It was also found that O3 exposure dramatically enhanced the proliferation of CD4-CD8- thymocytes stimulated by recombinant mouse interleukin-7 (rmIL-7), which is usually observed during the mammal aging process. Taken together, data conclude that short-term repetitive O3 exposure damages both innate and acquired immunity via altering the lymphocyte subset and cytokine profile, and via impact on thymocyte early development. O3-induced oxidative damage is one of the key factors leading to immune dysfunction in this mouse model.