Abstract: Alfalfa was used as the test material.The expression differences of RCI2 genes MsRCI2 A/2 B/2 Cderived from alfalfa were analyzed at 4 ℃,and then the genetic transformation of alfalfa was carried out by agrobacterium-mediated method.The effect of overexpression of MsRCI2 A/2 B/2 C gene on cold tolerance of alfalfa,in order to provide reference for cultivating cold tolerant alfalfa.The results showed that the MsRCI2 A/2 B/2 C genes were all induced by 4 ℃．The chlorophyll relative content,relative conductivity,and MDA content of the transgenic alfalfa changed significantly after12 days of 4℃treatment,indicating that their sensitivity to 4℃ was reduced.And after treatment at4℃,the transgenic plants accumulated more proline and soluble sugar than WT plants,and the activities of antioxidant enzymes（SOD,POD and CAT）increased rapidly at 6 days of treatment at4℃,and responded to cold stress more rapidly.In summary,the overexpression of MsRCI2 A/2 B/2 C genes improved the cold tolerance of alfalfa.