Abstract: The culture medium and fermentation conditions for the synthesis of astaxanthin from Rhodotorula glutinis RG-31 were studied by single factor experiment and orthogonal experiment. The results showed that the optimal culture medium of Rhodotorula glutinis RG-31 was glucose 10 g·L^-1, beef extract 3 g·L^-1, yeast powder 5 g·L^-1, K₂HPO₄ 1.5 g·L^-1、MgSO₄ 0.5 g·L^-1、CaCl₂ 0.2 g·L^-1. The optimal culture condition was the pH of 6, the culture temperature of 28℃, and the rotate speed of 240 r·min^-1. After culturing for 60 h, the content of astaxanthin was 7.41 μg·mL^-1, which was 2.61 times of that before optimization (2.84 μg·mL^-1), and the biomass was 20.5 g·L^-1, which was 1.90 times of that before optimization (10.8 g·L^-1).