Physiological factors for the production of pullulan by A. pullulans HP-2001 were optimized using orthogonal array method and their influences were compared using Qualitek-4 software. The analysis of variance (ANOVA) indicated that the most important factor for cell growth was yeast extract, whereas that for production of pullulan was glucose. The optimal conditions for cell growth were found to be 100.0 g/L glucose, 10.0 g/L yeast extract, and initial pH of 6.0, whereas those for the production of pullulan were 100.0 g/L glucose, 2.5 g/L yeast extract, and initial pH of 5.5. Among four mineral salts in the medium, potassium phosphate (K2HPO4) was found to be the most important factor for cell growth as well as production of pullulan. Next important salt for cell growth was (NH4)2SO4, whereas that for production of pullulan was NaCl. The optimal concentrations of K2HPO4, NaCl, MgSO4·7H2O, and (NH4)2SO4 for cell growth were 7.5, 1.00, 0.1, and 1.20 g/L, respectively, whereas those for production of pullulan were 2.5, 0.25, 0.8, and 0.30 g/L. The expected cell growth and the production of pullulan by A. pullulans HP-2001 under these optimized conditions were 12.61 and 11.49 g/L, respectively.