Abstract: In order to increase the viability of Bifidobacterium in microcapsules formation process and in human gastrointestinal system, the optimized additive amount of cryoprotectants complexed with xylo-oligosaccharides was determined by single-factor and orthogonal experimental test. The influences of optimal cryoprotectants on the properties of microcapsules were also studied in this paper. The results showed that the optimized cryoprotectants composition was 4% of xylo-oligosaccharides, 2% of glycerol, and 1% of sodium glutamate, which could achieve 81.5%±0.7% of embedding rate and 88.1%±0.3% of freeze-drying survival rate for Bifidobacterium in microcapsules. Compared to the blank group, the cryoprotectants adding group was increased 21.6% of final active bacteria loading rate. In addition, the surface of the microcapsules added with the cryoprotectants was smoother and denser than the blank group. In simulation human gastrointestinal system, after digested 2 h in artificial gastric juice, there were 65.9% of Bifidobacterium survived in cryoprotectants group, which increased 15.3% of survival rate comparing with blank group. The cryoprotectants also significantly improved the probiotic release from microcapsules in artificial intestinal fluid. After storage at 4 and 25 ℃ for 35 days, the number of survival Bifidobacterium in the microcapsules added with the cryoprotectants were 8.1 lg CFU/g and 7.0 lg CFU/g, which significantly (P<0.05) higher than the blank. Therefore, adding cryoprotectants complexed with xylo-oligosaccharides could improve the resistance of Bifidobacterium in adverse environments.