Candida krusei was osmotolerant yeast for the production of glycerol. Extracellular osmotic pressure was one of the key factors to induce the enzyme activity of glycerol-3-phosphate dehydrogenase(GPDH) which severely affected the glycerol productivity. Three osmoregulators such as NaCl, PEG4000 and glycerol were used to investigate their effects on yeast growth, glucose consumption and glycerol production. In order to determine the effect of extracellular glycerol concentration, different amounts of glycerol were initially supplemented as an osmoregulator to increase glycerol production. The maximum glycerol concentration attained 179g.L-1 with initial glycerol concentration of 80g.L-1 in medium, compared with 41g.L-1 in the control experiment. These results were successfully reproduced for fed-batch process in an air-lift reactor.