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Cholesterol is an important component of fresh biological baits and essential precursor to synthesize steroid hormones for crustacean. However, the cholesterol demand in Pacific white shrimp broodstock and its roles on ovary development and reproductive capacity are unknown. In the present study, female Pacific white shrimp broodstock (initial weight 51.3 ± 0.5g) were fed with three diets contained 0.5%, 1% or 1.5% cholesterol for 45 days. The results found that dietary cholesterol levels did not affect survival rate, weight gain, egg-laying number, vitellogenin content and vitellogenin receptor gene expression in the hepatopancreas. However, the hepatosomatic index, gonadosomatic index, egg diameter, vitellogenin level in the ovary and fertilized egg, C22:6n-3 (DHA) level in the fertilized egg, total protein, triglyceride and cholesterol contents were all increased in the 1.0% and 1.5% cholesterol groups than those in the 0.5% cholesterol group. Moreover, compared with the 0.5% and 1.0% cholesterol groups, the 1.5% cholesterol group had significantly higher gonad maturation number, mating rate, estradiol level in the hemolymph, steroid hormones synthesis related genes (HMGCR, StAR, 17βHSD and 1vMFBP) expression in the ovary and hepatopancreas. In conclusion, dietary 1.5% cholesterol could promote ovary development and reproductive capacity in female Litopenaeus vannameibroodstock by improving nutrients accumulation and steroid hormones synthesis pathway. Our results provide guidance for design of artificial compound feed and healthy development of shrimp broodstock farm.