Survival of bifidobacteria and preservation of their morphological characteristics after 12-month storage of lyophilized cells was studied for the strains of Bifidobacterium bifidum and B. animalis isolated and maintained in the microbial collection of the Department of Microbiology, Moscow State University. A combined approach to pre-lyophilization treatment of microorganisms and subsequent storage was developed in order to improve cell survival. Compared to the standard cryoprotector concentrations, sucrose and glucose (5% and higher) in skim milk, as well as freezing at ‒70°C with subsequent storage at the same temperature resulted in improved survival of bifidobacteria. Under such conditions, the number of viable cells (CFU) after 12 months of storage was two to three orders of magnitude higher than in the case of the standard lyophilization procedure. Investigation of dynamics of resistance of reactivated clones to such gastrointestinal stress factors as gastric juice and bile acids revealed preservation of these properties at all storage modes. However, since the number of surviving cells decreased during storage according to the standard procedure, the number of stress-affected cells was correspondingly lower. Reactivated cultures exhibited high resistance to oxygen, with survival decreasing to 35% of the initial level.