Folate-mediated one-carbon metabolism (FOCM) is closely associated with postharvest preservation. This study investigated the effects of exogenous nitric oxide (NO) on FOCM, storage quality, energy metabolism, and mitochondrial membrane integrity in cold-storage peach fruit. In this experiment, peaches were soaked with 1.5 mmol L−1S-nitrosoglutathione (GSNO) as NO donor, and the negative treatment (NT) solution containing 5 μmol L−1carboxy-PTIO (c-PTIO, NO scavenger), 200 μmol L−1NG-Nitro-L-arginine methyl ester (L-NAME, NO synthase-like enzyme inhibitor), and 200 μmol L−1sodium tungstate dihydrate (nitrate reductase inhibitor) and stored at 0°C. The results showed that NO decreased the activity ofS-adenosylmethionine synthase andS-adenosylhomocysteine hydrolase and increased the activity of methionine sulfoxide reductase A, as well as the content of N5-methyl-THF, the ratio of tetrahydrofolate (THF), homocysteine, methionine,S-adenosylmethionine (SAM), and SAM toS-adenosylhomocysteine compared with the control, indicating that NO effectively increased FOCM flux by affecting the activity of FOCM enzymes. Meanwhile, NO increased the activities of H+-ATPase, Ca2+-ATPase, cytochrome c oxidase, succinate dehydrogenase, and the contents of adenosine triphosphate and adenosine diphosphate, and maintained high energy charge in peaches during storage. NO retarded the increase in mitochondrial permeability transition, reactive oxygen species content, and the decrease in mitochondrial membrane fluidity, membrane potential, and swelling. NT treatment exhibited the opposite results. In conclusion, these results suggested that NO could induce the accumulation of folate and FOCM flux and maintain mitochondrial energy levels, which might be responsible for maintaining the quality of peaches during cold storage.