Abstract: This paper was aimed to search an optimal cryoprotectant for improving the cryopreservation quality of bovine mammary epithelial cells. The following 10 kinds of different ingredients of cryoprotectant were added to the fifth generation of BMECs respectively, group A: 85% DMEM +10% FBS +5% DMSO; group B: 80% DMEM +10% FBS +10% DMSO; group C: 75% DMEM +10% FBS +15% DMSO; group D: 70% DMEM +10% FBS +20% DMSO; group E: 85% DMEM +5% FBS +10% DMSO; group F: 75% DMEM +15% FBS +10% DMSO; group G: 70% DMEM +20% FBS +10% DMSO; group H: 80% DMEM +10% FBS +10% glycerol; group I: 70% DMEM +10% FBS +20 % glycerol; group J: 60% DMEM +10% FBS +30% glycerol, adjust the cell density to 1 × 106 /mL before cryopreservation.After the cell recovery , the survival and apoptosis rate of cells were calculated by trypan blue staining and PI/Hoechst33258 double staining respectively . Results indicated that the BMECs in these groups showed different levels of viability, morphology, and apoptosis after recovery. The vitality and 24 h adhering rate of group B and G was higher than that in other groups.Furthermore , the apoptosis rate of them were lower and there were no significant difference between them(P >0.05).After passage, the cells of group B grew the best.