Nitric oxide (NO) and reactive oxygen species (ROS) are two classes of key signaling molecules involved in various developmental processes and stress responses in plants. The burst of NO and ROS triggered by various stimuli activates downstream signaling pathways to cope with abiotic and biotic stresses. Emerging evidence suggests that the interplay of NO and ROS plays a critical role in regulating stress responses. However, the underpinning molecular mechanism remains poorly understood. Here, we show that NO positively regulates the activity of the Arabidopsis (Arabidopsis thaliana) cytosolic ascorbate peroxidase1 (APX1). We found that S-nitrosylation of APX1 at Cys32 enhances its enzymatic activity of scavenging H2O2, leading to the increased resistance to oxidative stress, whereas a substitution mutation at Cys32 causes the reduction of ascorbate peroxidase activity and abolishes its responsiveness to the NO-enhanced enzymatic activity. Moreover, S-nitrosylation of APX1 at Cys32 also plays an important role in regulating immune responses. These findings illustrate a novel mechanism by which NO regulates H2O2 homeostasis in plants, thereby establishing a molecular link between NO and ROS signaling pathways.