The objective was to investigate the effect of administering an additional dose of a PGF2α analog concurrent with the second GnRH (GnRH2) and at artificial insemination in the Ovsynch protocol on ovulation (OR) and pregnancy rates (PR) in Holstein lactating cows. Multiparous clinically healthy cows (parity: 3–5 lactation and 50 ± 5 days in milk (DIM)) were randomly allocated to three groups: Ovsynch (n = 23): GnRH (Alarelin acetate, 25 μg)-7 days- PGF2α (d-cloprostenol, 500 μg)-2 days-GnRH-16 to 18 h-FTAI plus 5 mL normal saline; OvsynchPG9 (n = 17): as Ovsynch plus administering another dose of PGF2α concurrent with GnRH2; and OvsynchPG10 (n = 28): as Ovsynch plus administering another dose of PGF2α at insemination. The ovaries of all selected animals were scanned by transrectal ultrasonography on days 9, 10, and 11 after the initiation (day 0) of the Ovsynch protocol to record the incidence of ovulation. Pregnancy diagnosis was performed by transrectal ultrasonography 50 ± 2 days after FTAI. The results showed that OR was greater in OvsynchPG9 (82.3%) than that in OvsynchPG10 (81.8%) or Ovsynch (81.2%), although the difference was not significant (P = 0.8205). In addition, PR/FTAI was greater in OvsynchPG9 (41.2%) than in Ovsynch (30.4%) and the difference was not significant (P = 0.8288). In conclusion, it was found that administering an additional dose of a PGF2α did not improve OR or PR in Ovsynch protocol in Holstein lactating cows.