Effects of silkworm pupae powder concentration, glucose concentration, cultural time and initial pH of culture medium on the fermentation of carotenoid were investigated. The results of single-factor experiments showed that when silkworm pupae powder concentration was 7.5 g/L, with glucose concentration at 30 g/L and cultural time of 4 d, as well as initial pH of culture medium of7.0, the carotenoid yield reached the maximum value of 14.51 mg/L, which was 46.9% higher than that of the control. In order to make further improvement on the carotenoid production, the orthogonal tests were performed and the results showed that the order of the factors affecting the carotenoid yield was as follows: cultural time>silkworm pupae powder concentration>glucose concentration>pH. The carotenoid yield reached the maximum value of 14.88 mg/L under the optimum fermentation conditions. Silkworm pupae powder concentration was 7.5 g/L, with glucose concentration at 30 g/L, and cultural time of 4 d, plus pH value at 6.0.