The rising atmospheric CO2 concentration profoundly impacts plant physiology and crop productivity, as elevated CO2 (eCO2) modulates stomatal property which in turn modulates plant photosynthesis, water use efficiency and plant defense responses. Extracellular reactive oxygen species (ROS) are required for both induction of stomatal closure and inhibition of stomatal opening, which are tightly controlled by different but interconnected mechanisms. However, current knowledge of the origin and regulatory role of ROS in stomatal CO2 responses is limited. In this study, a combinatorial approach using genetics and pharmacology indicates that both cell wall peroxidases and NADPH oxidases are required for apoplastic ROS production during eCO2-mediated stomatal closure, whereas eCO2-inhibited stomatal opening is dependent on NADPH oxidases but not on cell wall peroxidases, suggesting that the distinct source and regulatory role of apoplastic ROS are involved in stomatal movement induced by CO2. In addition, fully functional SA signaling pathway that is dependent on SHAM-sensitive peroxidases is required for eCO2-mediated stomatal closure but not for eCO2-inhibition of stomatal opening. We have provided further evidence that stomatal CO2 responses that are triggered by various plant hormones are differentially regulated by distinct sources of elicited ROS in Arabidopsis.