The present study was conducted to investigate the effects of administering estradiol (estradiol-17β, 2.5 mg) plus progesterone (P4, 50 mg) as a part of a controlled internal drug release (CIDR)-based superovulation protocol on ovarian follicles development, and oocyte recovery as well as on in vitro development in prepubertal calves after follicle stimulating hormone (FSH) stimulation. Calves in the treatment group exhibited significantly decreased (P < 0.05) numbers of both small and large antral follicles compared to the control group A and B (<3 mm in diameter [small], 3.4 versus 12.8, 11.6; > 7 mm in diameter [large], 0 versus 7.6, 7.2). The treated animals also exhibited an increased (P < 0.05) proportion of usable oocytes from the total oocytes recovered compared with those of the control group A and B (77% versus 50.2%, 47.8%). Moreover, the rate of cleavage and the percentage of blastocysts displayed an increased trend in the treatment group compared to controls A and B (59.2% versus 50.5%, 50.8%; 15% versus 12.4%, 11.6%, respectively). In conclusion, the application of estradiol-17β plus P4 at CIDR insertion prior to FSH stimulation affected ovarian follicles development and exerted a beneficial effect on the in vitro development of calf oocytes.