© 2021, University of Veterinary and Pharmaceutical Sciences. All rights reserved.This study focused on determining the success of inducing endogenous progesterone source (by formation of accessory corpus luteum) by human chorionic gonadotropin (hCG) administration on day 4 post timed artificial insemination (TAI) and on evaluating the effect of the day of hCG administration (day 4 or 5 post TAI) on pregnancy rates in repeat breeder cows according to their responses to hCG administrations. Non-pregnant cyclic lactating Holstein dairy cows with more than three services despite no clinical reproductive abnormalities were enrolled in the study. All cows (n = 192) received the Ovsynch protocol (GnRH-7d-PGF2α-56h-GnRH-16/18h-TAI) and hCG administration (1500 IU, i.m.) was performed in group hCG4 (n = 64) on day 4 and in group hCG5 (n = 68) on day 5 post TAI. The control group (CON, n = 60) did not receive any hCG treatment. The responses (ovulation rate) to hCG (~64%) were similar on days 4 and 5 post TAI. Pregnancy per AI (P/AI) at 31 days was similar among the groups (48.3% in CON, 48.4% in hCG4, and 42.6% in hCG5). Regardless of the day of the treatment (hCG4 + hCG5), the cows had an ovulatory response to hCG (54.1%), and a greater P/AI (P = 0.007) at 31 days than those without a response (29.8%). The administration of hCG on either day 4 or 5 post TAI did not increase overall pregnancy rates, however, the ovulatory response to the post TAI hCG administration on day 4 improved fertility in repeat breeder cows. Further studies are needed with a larger number of animals to reveal this potentiallly beneficial effect.