Our objective was to evaluate the effect of decreasing progesterone (P4) before initiation of an Ovsynch protocol on ovulatory response to the first GnRH injection (G1) and pregnancies per AI (P/AI). Lactating Holstein cows (n = 800) were synchronized using a Double Ovsynch protocol [Pre-Ovsynch protocol (GnRH; 7 d, PGF_2α; 3 d, GnRH) followed 7 d later by an Ovsynch-56 protocol (G1; 7 d PGF_2α; 24 h, PGF_2α; 32 h, GnRH)] to receive first timed artificial insemination (TAI; 80 ± 3 DIM) 16 h after the last GnRH treatment. Cows were randomly assigned to receive a half-dose of PGF_2α (12.5 mg dinoprost tromethamine) 2 d before G1 (TRT) or serve as untreated controls (CON). Data were analyzed by logistic regression using GLIMMIX and ANOVA with MIXED procedures of SAS. Overall, CON cows had greater (P < 0.01) P4 than TRT cows at G1 (4.2 vs. 2.1 ng/mL). Ovulatory response to G1 was greater (P < 0.01) for TRT vs. CON cows [81.9% (90/110) vs. 60.9% (70/115), respectively]. Luteal regression during the second Ovsynch protocol did not differ (P = 0.33) between treatments [15.2% (15/99) vs. 10.6% (11/104); TRT vs. CON]. At 32 d after TAI, P/AI did not differ (P = 0.34) between treatments [56.2% (223/397) vs. 52.8% (209/396); TRT vs. CON]. At 67 d after AI, P/AI also did not differ (P = 0.56) between treatments [50.8% (190/374) vs. 48.6% (179/368); TRT vs. CON]. Pregnancy loss from 32 to 67 d after TAI did not differ [10.0% (21/211) vs. 9.6% (19/198); TRT vs. CON; P = 0.90]. Overall, cows that ovulated to G1 had greater (P = 0.02) P/AI compared with cows that did not ovulate [58.2% (89/153) vs. 41.5% (27/65), respectively]. The increase in P/AI in ovulating cows (16.7%) and observed increase in ovulation (21%; TRT – CON) produced an expected increase of 3.5% in P/AI in TRT vs. CON; similar to the observed 3.4% difference. Thus, administration of a half-dose of PGF_2α 2 d before G1 during a Double Ovsynch protocol decreased P4 at G1 and increased ovulatory response. Larger studies are needed to determine if this modified protocol increases P/AI. Supported by USDA Hatch project 231440.