In this study, the mechanism of cold stress tolerance in cucumber fruits pretreated with folic acid was investigated. Control group and the group treated with 5 mgL-1 folic acid were stored for 15 days at 4 °-C. The results showed that, in comparison with the control, treatment with folic acid resulted in reduced chilling injury and decreased electrolyte leakage. The cucumber fruits treated with folic acid showed higher chlorophyll contents in storage conditions with suppressed chlorophyllase enzyme activity. Exogenous folic acid treatment also increased the activity of arginine decarboxylase and ornithine decarboxylase resulting in the accumulation of polyamine contents. Also, higher levels of proline were observed in the fruits treated with folic acid, which is attributed to the increased activity of proline synthesizing enzymes △1-pyrroline-5-carboxylate syntheses and ornithine aminotransferase and also reduced activity of proline dehydrogenase enzyme that decompose proline. Generally, the results showed that folic acid treatment increased the resistance to the cold stress by regulating the activity of enzymes involved in the biosynthesis of proline, chlorophyll, and polyamines.