We evaluated a simple, short hormonal protocol comprising follicular wave synchronization using a progesterone-releasing device and estradiol benzoate 5 days before ovum pick-up (OPU) followed by a single follicle-stimulating hormone injection 2 days before OPU to improve oocyte quality and outcomes of in vitro embryo production (IVP) and embryo transfer (ET) in Japanese Black cows. A total of 12 cows were used in this study, with 6 cows in each of the Treatment and Non-Treatment groups. The proportion of good-quality oocytes, transferable blastocysts per OPU session, and pregnancy rate following ET of IVP-derived blastocysts were all higher in the Treatment group than in the Non-Treatment group (63.0 ± 7.7% vs. 47.5 ± 7.6%, 4.0 ± 0.7 vs. 2.6 ± 0.5, and 55.6% vs. 19.0%, respectively; P < 0.05). These results demonstrate that the present protocol, which requires only two procedures before OPU, effectively improves OPU-IVP and ET outcomes in Japanese Black cows.