The capacity of lipid and carotenoid production by Rhodotorula glutinis was investigated under different irradiation conditions, temperatures and C/N ratios. The results showed that dark/low-temperature could enhance lipid content, while irradiation/high-temperature increased the yields of biomass and carotenoid. The optimum C/N ratio for production was between 80 and 100. A two-stage cultivation strategy was used for lipid and carotenoid production in a 5 L fermenter. In the first stage, the maximum biomass reached 28.1 g/L under irradiation/high-temperature. Then, the cultivation condition was changed to dark/low-temperature, and C/N ratio was adjusted to 90. After the second stage, the biomass, lipid content and carotenoid reached 86.2 g/L, 26.7% and 4.2 mg/L, respectively. More significantly, the yields of biomass and lipid were 43.1% and 11.5%, respectively. Lipids contained 79.7% 18C and 16.8% 16C fatty acids by GC analysis. HPLC quantified the main carotenoids were beta-carotene (68.4%), torularhodin (21.5%) and torulene (10.1%). Crown Copyright (C) 2014 Published by Elsevier Ltd. All rights reserved.