The objective of this randomized controlled experiment was to evaluate the effect on first service reproductive outcomes for lactating Holstein cows of a targeted reproductive management program including insemination in estrus (AIE) after a modified Double-Ovsynch protocol and re-enrollment of non-estrus cows in a synchronization of ovulation protocol. Lactating dairy cows (n = 2,101) from a commercial dairy farm fitted with neck-attached sensors for automated detection of estrus were synchronized with a Double-Ovsynch protocol up to the first PGF2α (PGF-L) of the Breeding-Ovsynch portion of the protocol (GnRH, 7 d later PGF2α, 3 d later GnRH, 7 d later GnRH, 7 d later PGF2α, 1 d later PGF2α). At PGF-L, cows were blocked by parity and type of semen used for first service and then randomly assigned to the DO-G80 (n = 1,048) and the DO-ED-P4Ov (n = 1,053) treatments. Cows in the DO-G80 treatment received GnRH for induction of ovulation at 80 h after induction of luteolysis [GnRH 80 h after PGF-L, 16 h later timed AI (TAI)]. All cows with automated estrus alerts (AEA) after PGF-L and before the GnRH treatment received AIE. Cows in the DO-ED-P4Ov treatment were eligible to receive AIE for 7 d after PGF-L and if not AIE were submitted to a modified Ovsynch protocol with progesterone (P4) supplementation (P4-Ov: GnRH + P4 device in, 7 d later PGF2α and P4 device removal, 1 d later PGF2α, 32 h later GnRH, 16 h later TAI). Data were analyzed by logistic regression and ANOVA. The DO-ED-P4Ov treatment increased the proportion of cows AIE (+41%; DO-G80 46.7% and DO-ED-P4Ov 88.1%) and first service P/AI (+8%) compared with the DO-G80 treatment through enhanced P/AI of primiparous cows (primiparous DO-G80 50.5% and DO-ED-P4Ov 63.6%; multiparous DO-G80 45.3% and DO-ED-P4Ov 48.5%) and P/AI in the range of 45% for non-estrus cows targeted with the P4-Ovsynch protocol. Despite increased costs of individual AI services, the DO-ED-P4Ov treatment also had reduced costs per pregnancy. In conclusion, a targeted reproductive management (TRM) program designed to increase fertility through insemination of cows in estrus after a modified Double-Ovsynch protocol and a targeted intervention for non-estrus cows might be an alternative to increase first service fertility and reduce costs per pregnancy primarily through better performance of primiparous cows. This program could also have other herd management benefits including identifying cows of different fertility potential and the distribution of AI services over more days of the week compared to TAI.