The objective of this study was to investigate the effect of GnRH and EB injection on the SOV response in early postpartum suckling Japanese Black cows. Forty-eight cows were included in this study. All cows received a controlled internal-drug-releasing device containing 1.9 g progesterone (CIDR; Easi-Breed; InterAg, Hamilton, New Zealand) at 40 days postpartum, and were subsequently superstimulated with a total dose of 20 armour units FSH (Antrin 40; Kawasaki-Mitaka, Kanagawa, Japan) twice daily, with gradually decreasing doses from day 45 till day 47. CIDR was withdrawn with 0.75 mg of cloprostenol (Estrumate; Nagase Medicals, Itami, Japan) injection at day 47. After FSH treatment, the cows were assigned to 3 groups: (1) 100 μg GnRH (Conceral; Nagase Medicals) injection at day 49 (GnRH, n = 19), (2) 2 mg EB (Kawasaki-Mitaka) injection at day 48 (EB, n = 15), and (3) control (n = 14). AI was carried out twice based on the estrous behavior of the cows, which was examined several times daily. Embryos were nonsurgically collected 7 to 8 days after estrus. The ovaries were examined by ultrasonography, and the number of CL and unovulated follicles (UF) were counted. After uterine flushing, the cows were re-employed for reproductive purposes. The intervals to first estrus after flushing and days open were examined. Blood samples were collected temporally, and concentrations of progesterone (P4) and estradiol (E2) were measured. The ovaries were examined by ultrasonography and the number of CL, small follicles (<5 mm in diameter), medium follicles (between 5 and 10 mm in diameter), and large follicles (>10 mm in diameter) were counted temporally. Data were analyzed by GLM and Dunnett’s T3 test. There were no significant differences between both treatment groups and the control group in the number of CL (15.2 ± 6.8, 18.9 ± 9.4, and 14.9 ± 8.9 in the GnRH, EB, and control groups, respectively; mean ± SD), UF (5.3 ± 3.2, 4.7 ± 3.2, and 7.2 ± 9.5, respectively), recovered ova or embryos (11.4 ± 8.1, 11.1 ± 8.9, and 11.1 ± 9.1, respectively), transferable embryos (6.8 ± 5.3, 7.5 ± 7.6, 7.2 ± 6.7, respectively), and freezable embryos (6.3 ± 5.6, 6.6 ± 7.9, and 5.7 ± 6.1, respectively). Furthermore, there were no significant differences in the intervals to first estrus after flushing and days open (12.7 ± 13.6 and 90.0 ± 23.1 in the GnRH, 8.6 ± 5.3 and 79.1 ± 20.1 in the EB, and 9.4 ± 6.0 and 88.5 ± 33.8 in the control groups, respectively). Changes in the P4 level were similar among the 3 groups. The E2 level of the EB group at 15 h after EB injection was significantly higher (P < 0.05) than that in the other groups (19.0 ± 15.7, 49.4 ± 22.8, and 8.0 ± 5.7 pg mL–1 in the GnRH, EB, and control groups, respectively). The number of small follicles in the control group (17.0 ± 12.2) was significantly greater (P < 0.05) than that in the GnRH group (7.4 ± 7.2) at the first AI. These results suggest that EB and GnRH injection after SOV treatment in early postpartum Japanese Black cows does not affect the SOV response or embryo quality.