In this study, pullulan production was achieved by whole-cell bioconversion with Aureobasidium pullulans CCTCC M 2012259. Response surface methodology was applied to optimize the seed medium for incubating cells with high capability of pullulan bioconversion. Three medium components, namely, yeast extract, MgSO4·7H2O, and glucose were identified by Plackett-Berman design as significant factors affecting the cells' pullulan bioconversion capability. A three-level Box-Behnken design was then employed to determine the optimal levels of the three components. A mathematical model was developed to show the influence of each medium component and its effects on the cells' pullulan bioconversion capability. The model predicted a maximum pullulan bioconversion capability of 32.28 mg/g/h at the optimal yeast extract, MgSO4·7H2O, and glucose concentrations of 3.57, 0.18, and 63.97 g/l, respectively. The validation experiments showed that the cells' pullulan bioconversion capability was improved by 23.1% when the optimal medium was used, as compared with that obtained with the basic medium. Subsequently, the gene expression and activities of the key enzymes involved in pullulan biosynthesis were evaluated. When the optimal medium was employed, the transcriptional levels of pgm1 and fks were up-regulated by 2.5- and 1.2-fold, respectively, and the α-phosphoglucose mutase and glucosyltransferase activities were increased by 17 and 19%, respectively, when compared with those achieved using the basic medium. These results indicated that pullulan bioconversion using A. pullulans CCTCC M 2012259 as the whole-cell catalyst is an attractive approach for efficient pullulan production and can be applied for the production of other polysaccharides.