Purpose: To investigate the effect of vitamin B2 (VB2) on ovarian development during puberty. Methods: Four groups of domestic hens (Jinghong-1 strain, 12 hens/group) were housed under standard conditions and fed basal diet with or without graded doses of VB2 (10-40 mg/kg). At 10 weeks old, 9 hens were sacrificed from each group. Plasma levels of AST, ALT, steroid hormones and growth hormones were determined. In addition, some essential mineral elements in the ovarian tissue of the hens were assayed. Results: Treatment with VB2 significantly improved ovary and liver organ indices (p < 0.01), but had no deleterious effect on the liver. The different doses of VB2 exerted regulatory effects on homeostasis of essential elements in the ovary (p < 0.01). Moreover, VB2 treatment elevated plasma levels of progesterone (PR) and estrogen (ES), suggesting that it might regulate steroid hormone levels. Conclusion: These results indicate that VB2 enhances the development of the ovaries during puberty.