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Objective: To evaluate the development of ovarian follicles in female albino rats following vitamin D3 supplementation. Methods: Eighteen prepubertal female albino rats, aged 3-4 weeks, weighing (70.25±9.16) g, were assigned to three groups (n=6 in each group). Group A was treated with 5.00 mL/kg of distilled water and served as the control group, group B was treated with 0.025 mg/kg of vitamin D3 dissolved in distilled water, and group C was treated with 0.125 mg/kg of vitamin D3 dissolved in distilled water. All treatments were administered orally, twice weekly for 28 days. Blood and ovaries were harvested under anaesthesia. Serum vitamin D3 levels were determined by using spectrophotometric method. Ovaries were processed for histology and every10th hematoxylin and eosin stained-section was selected for histomorphometry. The number of follicles at each developmental stage was estimated. Results: Both 0.025 mg/kg and 0.125 mg/kg of vitamin D3 significantly increased serum concentrations of vitamin D3 and calcium (P0.05). The control group had fewer growing follicles (primary, secondary and antral follicles) and more non-growing follicles (primordial and atretic follicles) when compared with the vitamin D3-supplemented groups (P<0.05). Vitamin D3 at 0.025 mg/kg significantly increased antral follicles and corpora lutea counts (P<0.05). Vitamin D3 at 0.125 mg/kg significantly increased total, primordial and atretic follicles counts (P<0.05), but significantly decreased primary, secondary, antral follicles count, ovarian weight, relative ovarian weight, and ovarian surface area when compared with the control group and rats treated with 0.025 mg/kg of vitamin D3 (P<0.05). Conclusions: Vitamin D3 supplementation at 0.025 mg/kg can enhance optimal ovarian follicle recruitment and development in female rats.