Melatonin has been reported to alleviate chilling symptoms in postharvest peach fruit during cold storage, however, the mechanism involved is largely unknown. To better understand its role in chilling tolerance, here we investigated the effects of melatonin on oxidative damage in peach fruit subjected to chilling after harvest. Chilling injury of peaches was dramatically reduced by melatonin treatment. Melatonin induced hydrogen peroxide (H₂O₂) content at the early stage of storage but inhibited its accumulation thereafter. Meanwhile, melatonin also up-regulated the expression of genes involved in antioxidant responses in peaches. In addition, compared to the control fruit, peaches treated with melatonin displayed higher transcript abundance of ascorbic acid (AsA) biosynthetic genes and consequently increased the AsA content. Our results suggested that in response to melatonin during chilling, the high H₂O₂ level in the treated peaches at the initial time of storage, may work as a signaling molecule to induce protective mechanisms via up-regulating the expression of antioxidative genes and increasing AsA content. On the other hand, after the transient increase in the treated peaches, H₂O₂ was efficiently removed because of the activated antioxidant systems, which was associated with the higher chilling tolerance induced by melatonin.