Chestnut (Castanea mollissima Bl.) is a popular nut in Chinese and International market. However, storage of chestnut is difficult. In this study, the relationships of respiration rate, metabolic pathway, key enzymatic activities, metabolites, and membrane peroxidation with chestnut rotting during storage at room temperature (25 °C) and low temperature (0–3 °C) were investigated. The results show that the respiration rate of chestnut gradually decreases during storage at room temperature. Under the lower temperature, the respiration rate reduced 67.53% within 1 day, then gradually increased. Compared with room temperature, the lower temperature also significantly inhibited glycolysis, maintained higher level of tricarboxylic acid cycling, and reduced the conversion of pyruvate to ethanol and lactate. In addition, chloramphenicol acetyltransferase activities were inhibited while superoxide dismutase activities were high under the lower temperature, which resulted in the reduction of membrane peroxidation. Subsequently, the chestnut rotting rate was significantly reduced. In conclusion, the primary reason for chestnut rotting is the reduction of proper physiologic function and then bacteria and pathogen infection. Chestnut rotting rate during storage can be significantly reduced through an appropriate environment and new techniques and chemicals.