The high carotenoid content in docosahexaenoic acid (DHA) oil may serve as an avenue to avoid DHA oxidation during storage. To obtain carotenoid-rich DHA oil from Schizochytrium sp. S31, the effects of several key medium components were investigated. Shake-flask experiment revealed that in optimal conditions, glycerol was used as the carbon source with 0.25 g/L of KH2PO4 and a C/N ratio of 75:20. Fed-batch cultivation further showed that a high lipid accumulation (30.81 g/L) with 497.56 mu g/g carotenoid and 12.24 g/L DHA was obtained under the optimized conditions. The increased carotenoid content resulted in improved defense against environmental stress, as indicated by the catalase (CAT) and superoxide dismutase (SOD) activities. Further transcriptome analysis demonstrated that the synthesis pathway of beta-carotene was enhanced during the lipid turnover stage, which suggested that more carotenoids were produced to reduce oxidative stress. This study presented an efficient strategy for DHA oil production with high carotenoid content.