Our objectives were to test the efficacy of intravaginal (IVG) administration of PGF_2α (PGF) to induce corpus luteum (CL) regression, compare circulating progesterone (P4) profiles in cows receiving IVG versus intramuscular treatment with PGF, and evaluate reproductive outcomes. Lactating Holstein cows were synchronized using a Double-Ovsynch (DO) protocol (GnRH-7d-PGF-3d-GnRH-7d-GnRH-7d-PGF-56h-GnRH-16h-AI) to receive timed AI (TAI) at 67 ± 3 DIM. Seven days after the first GnRH treatment (time 0), cows with at least one visible CL ≥ 15 mm were blocked by parity and randomly assigned to a control (CON; n = 201) or treatment group (TRT; n = 201). Cows in CON received 25 mg of PGF (Dinoprost tromethamine) intramuscular. Cows in TRT received 2 25 mg doses of PGF 12 h apart delivered through a catheter in the cranial portion of the vagina. Blood samples were collected at 0, 12, 48 and 72 h after treatment. Ovulation to the first GnRH of DO was determined through transrectal ultrasonography. Only cows with P4 ≥ 1 ng/mL (functional CL) at time 0 (CON = 169; TRT = 179) were included in the analyses. Binomial and quantitative data were analyzed by logistic regression and ANOVA with repeated measures, respectively. Concentrations of P4 and the proportion of cows with a new CL at time 0 did not differ (P > 0.10). Overall, the proportion of cows with CL regression using 1 ng/mL (P = 0.48; CON = 87.0%, TRT = 84.4%) or 0.5 ng/mL (P = 0.98; CON = 81.1%, TRT = 81.0%) of P4 as cutoff did not differ. Concentrations of P4 were affected by treatment (P = 0.01), time (P < 0.001) and the treatment by time interaction (P < 0.001). Cows in TRT had greater (P < 0.001) mean P4 at 12 h than cows in CON. Mean P4 did not differ (P > 0.10) at 48 or 72 h after treatment. The proportion of cows with estrus recorded within 3 d of treatment (P > 0.10; CON = 47.0%; TRT = 51.1%), ovulation risk after treatment (CON = 88.0%; TRT = 84.1%) and pregnancy per AI after TAI (CON = 49.7%; TRT = 56.3%) did not differ. We concluded that 2 intravaginal doses of 25 mg of PGF 12 h apart were as effective as a single 25 mg intramuscular dose of PGF for inducing luteal regression in lactating dairy cattle. Funded by USDA-NIFA under NYC1007421 and NYC 1014955.