Abstract: Main factors affecting production of glycerol in shake flasks in a process of repeated batch fermentation were studied. The experiments indicated that optimal concentration of corn steep liquor in repeated batch fermentation was lower than that in conventional batch fermentation because of higher cell density. The favorable occasion for reuse is when residual glucose concentration decreased to about 10g·L ^-1 ,and cell viability was about 60%.The residual harmful metabolite among centrifuged cells that were not washed inhibited glycerol production in the process of repeated batch fermentation, however, washing cells before centrifuged cells were reused would eliminate inhibition.