Abstract Vitamin D receptors and vitamin D3-metabolizing enzymes have been found to be highly expressed in the ovaries and spermatophores of fish. However, the role of vitamin D3 on fish gonadal development has rarely been reported. In this study, 2-month-old female zebrafish were fed with different concentrations of vitamin D3 diets (0, 700, 1400, and 11 200 IU/kg) to investigate the effects of vitamin D3 on ovarian development. The diet with 0 IU/kg vitamin D3 resulted in elevated interstitial spaces, follicular atresia, and reproductive toxicity in zebrafish ovaries. Supplementation with 700 and 1400 IU/kg of vitamin D3 significantly increased the oocyte maturation rate; upregulated ovarian gonadal steroid hormone synthesis capacity; and elevated plasma estradiol, testosterone, and ovarian vitellogenin levels. Furthermore, the current study identified a vitamin D response element in the cyp19a1a promoter and demonstrated that 1.25(OH)2D3-vitamin D response directly activated cyp19a1a production through activating the vitamin D response element. In conclusion, this study shows that an appropriate concentration of vitamin D3 can promote zebrafish ovarian development and affect vitellogenin synthesis through the vdr/cyp19a1a/er/vtg gene axis.