Abstract: Sequential methodology combining a screening stage by fractional factorial design and an optimization stage by central composite design was applied to enhance canthaxanthin production of Dietzia natronolimnaea HS-1 in shake flask cultures. Five variables (pH, luminous intensity, inoculum percent, concentration of glucose and concentration of NaCl) were studied with the first design and the results revealed that three factors (pH, concentration of glucose and concentration of NaCl) had greater influence on the canthaxanthin production (p<0.01). A central composite design was then used in the second step to determine the maximum canthaxanthin concentration. The optimum condition for the highest canthaxanthin production (5.32 mg L-1) was a pH of 7.53, glucose concentration of 25.90 g L-1 and NaCl concentration of 3.42 g L-1.