Hydrogen sulfide (H2S) has been identified as an important gaseous signal in plants. Here, we investigated the mechanism of H2S in alleviating postharvest senescence and rotting of Kyoho grape. Exogenous application of H2S released from 1.0 mM NaHS remarkably decreased the rotting and threshing rate of grape berries. H2S application also prevented the weight loss in grape clusters and inhibited the decreases in firmness, soluble solids, and titratable acidity in grape pulp during postharvest storage. The data of chlorophyll and carotenoid content suggested the role of H2S in preventing chlorophyll breakdown and carotenoid accumulation in both grape rachis and pulp. In comparison to water control, exogenous H2S application maintained significantly higher levels of ascorbic acid and flavonoid and total phenolics and reducing sugar and soluble protein in grape pulp. Meanwhile, H2S significantly reduced the accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2 (∙-)) in grape pulp. Further investigations showed that H2S enhanced the activities of antioxidant enzymes ascorbate peroxidase (APX) and catalase (CAT) and decreased those of lipoxygenase (LOX) in both grape peels and pulp. In all, we provided strong evidence that H2S effectively alleviated postharvest senescence and rotting of Kyoho grape by modulating antioxidant enzymes and attenuating lipid peroxidation.