Aims: Carbon monoxide (CO) confers antiproliferative effects on T cells; however, how these effects are produced remains unclear. Reactive oxygen species (ROS) have recently emerged as important modulators of T cell proliferation. In this study, we aimed to determine whether the inhibitory effects of CO on T cell proliferation are dependent on the inhibition of ROS signaling. Results: Pretreatment with CO-releasing molecule-2 (CORM-2) had potent inhibitory effects on mouse T cell proliferation stimulated by anti-CD3/CD28 antibodies. Interestingly, CORM-2 pretreatment markedly suppressed intracellular ROS generation as well as the activity of NADPH oxidase and mitochondrial complexes I-IV in T cells after stimulation. The inhibitory effects of CORM-2 on both ROS production and T cell proliferation were comparable with those produced by the use of antioxidant N-acetylcysteine or a combined administration of mitochondrial complex I-IV inhibitors. Moreover, increasing intracellular ROS via hydrogen peroxide supplementation largely reversed the inhibitory effect of CORM-2 on the proliferation of T cells. The inhibitory effects of CORM-2 on both cell proliferation and intracellular ROS production were also shown in a T cell proliferation model involving stimulation by allogeneic dendritic cells or phorbol 12-myristate 13-actetate/ionomycin, as well as in spontaneous cell proliferation models in EL-4 and RAW264.7 cells. In addition, CORM-2 treatment significantly inhibited T cell activation in vivo and attenuated concanavalin A-induced autoimmune hepatitis. Innovation: CO inhibits T cell proliferation via suppression of intracellular ROS production. Conclusion: The study could supply a general mechanism to explain the inhibitory effects of CO on T cell activation and proliferation, favoring its future application in T cell-mediated diseases.