Abstract: An optimal two-stage temperature control strategy was established to improve the yield and the productivity of glutathione in the batch fermentation, according to the effects of temperature on cell metabolism. Through single-factor experiments, the optimum growth temperature of Hansenula polymorpha DL-1, the optimum biosynthesis temperature of GSH and initial pH value of medium were obtained. The result showed that the effect of temperature on glutathione production was different from that on cell growth and maximum glutathione was obtained at 37℃, but the optimum growth temperature was at 30℃. When the temperature shifted from 45℃to 37℃for further cultivation of 16h, a glutathione yield of 420.16mg/L and an intracellular glutathione content of 105.19mg/g were achieved, which was 2.34 and 1.27 times than that of the constant temperature fermentation, respectively. Subsequently, scale-up studies were conducted in a 5L fermenter, a cell dry weight of 42.35g/L and a glutathione content of 927.68mg/L were achieved. Therefore, the temperature-shift strategy would be a valuable alternative to improve glutathione production.