To improve the performance of cross-flow membrane filtration of pullulan broth from Aureobasidium pullulans, the effect of the cultivation conditions was examined. In particular, the sucrose concentration in the medium was changed over a wide range. By decreasing the sucrose concentration the distribution of morphology of the microbial cells in the broth changed; the yeast-like form became predominant and, as a result, the specific resistance of the microbial cake was lowered. When the broth was fermented with a sucrose concentration of 2.5% or lower, the filtration characteristics were greatly improved by periodic closure of permeation during cross-flow filtration.