To evaluate an analog library of betaine-type cellular metabolites, which are naturally found in polar fish for survival in subzero temperatures, for preventing denaturation of enzymes during freezing. Comparison of the cryoprotective ability of reported cryoprotectants, such as dimethylsulfoxide, glycerol, ectoine, hydroxyectoine, and trehalose, with betaine-type analogs using alpha-glucosidase revealed that analogs introducing C-3-C-6 alkyl chains into an ammonium cation retained 20 % higher activity than the control cryoprotectants at the same concentration. In particular, the analog possessing triplicate n-butyl chains showed a profound effect. It allowed retention of enzyme activity to 95 % even after 100 freeze-thaw cycles, while addition of the control cryoprotectants decreased the activity to 10-20 %. The cryoprotective ability of betaine-type analogs can be applied not only to alpha-glucosidase but also other enzymes such as beta-glucosidase, alkaline phosphatase, lactose dehydrogenase, sulfatase, and horseradish peroxidase. Synthetic betaine-type metabolite analogs possess practicable cryoprotective ability for various enzymes, and are considerably superior to previously reported cryoprotectants.