A number of recent studies identified hydrogen sulfide (H S) as an important signal in plant development and adaptation to environmental stress. H S has been proven to participate in ethylene-induced stomatal closure, but how the signaling pathways of H S and ethylene interact is still unclear. Here, we reveal how H S controls the feedback-regulation of ethylene biosynthesis in tomato ( ) under osmotic stress. We found that ethylene induced the production of H S in guard cells. The supply of hypotaurine (HT; a H S scavenger) or DL-pro-pargylglycine (PAG; a synthetic inhibitor of H S) removed the effect of ethylene or osmotic stress on stomatal closure. This suggests that ethylene-induced H S is a downstream component of osmotic stress signaling, which is required for ethylene-induced stomatal closure under osmotic stress. We further found that H S inhibited ethylene synthesis through inhibiting the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidases (ACOs) by persulfidation. A modified biotin-switch method (MBST) showed that H S can induce persulfidation of LeACO1 and LeACO2 in a dose-dependent manner, and that persulfidation inhibits the activity of LeACO1 and LeACO2. We also found that LeACO1 is persulfidated at cysteine 60. These data suggested that ethylene-induced H S negatively regulates ethylene biosynthesis by persulfidation of LeACOs. In addition, H S was also found to inhibit the expression of genes. The results provide insight on the general mode of action of H S and contribute to a better understanding of a plant's response to osmotic stress.