Reactive oxygen species (ROS) and nitric oxide (NO) are two critical classes of signaling molecules that regulate plant development and stress responses. The intracellular level of S-nitrosoglutathione (GSNO), a major bioactive NO species, is regulated by the highly conserved GSNO reductase (GSNOR). However, the molecular mechanisms underlying ROS-mediated regulation of GSNOR remain largely unclear. Here, we show that H2O2 negatively regulates the activity of GSNOR1 during ovule development in Arabidopsis. S-sulfenylation of GSNOR1 at Cys-284 inhibits its enzymatic activity. A GSNOR1C284S mutation causes a reduction of the total SNO level in pistils, thereby disrupting NO homeostasis and eventually leading to defective ovule development. These findings illustrate a unique mechanism by which ROS regulates ovule development through S-sulfenylation-mediated inhibition of the GSNOR activity, thereby establishing a molecular link between ROS and NO signaling pathways in reproductive development.