【Objective】To analyze the effects of melatonin (MT) on the growth, biochemical characteristics and expression of cold-responsive related genes in Grapevine suspension cells under low-temperature stress, to screen for the appropriate MT concentration, and to investigate the mechanism of action of MT in enhancing the cold tolerance of Grapevine.【Methods】Using ''Pinot noir'' Grapevine suspension cells as test material, we investigated the effects of different concentrations of MT [0 (CK), 50 (T1), 100 (T2), 200 (T3), 300 μmol/L (T4)] on the growth of the suspension cells, the content of malondialdehyde (MDA), the content of proline (Pro) and the expression of genes related to cold-responsive genes, at a low-temperature (4 ℃) stress, Proline (Pro) content, antioxidant enzyme activities, and the trends of gene expression changes related to cold response to T2 treatment. 【Results】(1) Under low-temperature stress, the dry mass of cells in each MT treatment showed a trend of increasing then decreasing and finally stabilizing or decreasing during the culture cycle, and was significantly higher than that of CK at the end of the treatment period.(2) The MDA and Pro contents of each treatment firstly increased and then decreased with the time of treatment, and reached the peak at 24 h or 48 h. The MDA contents of each MT treatment were significantly lower than that of CK, and the Pro content was significantly higher than that of CK, and all of them were significantly higher than that of CK, and were in the same order. The MDA content of each MT treatment was significantly lower than that of CK, while the Pro content was significantly higher than that of CK, with T2 treatment being the lowest or the highest; (3) the antioxidant enzyme activities of each MT treatment were significantly higher than that of CK during the same period, with T2 treatment being the highest; with the prolongation of time, the activities of POD and CAT of each treatment firstly rose and then fell, while the activities of SOD and APX firstly fell and then rose, and reached the highest at 24 or 6 h, respectively. (4) The relative expression of cold-responsive genes VvCBFs, VvICE1, VvRD29B and VvKIN-2 in T2-treated suspension cells were all up-regulated significantly at different times compared with the corresponding CK.【Conclusion】Under low-temperature stress, the appropriate concentration (100 μmol/L) of MT could increase the relative expression of antioxidant enzymes and cold-response-related genes, increase the Pro content, decrease the MDA content, and ultimately enhance the low-temperature tolerance of Grapevine suspension cells.