In previous work, using porcine luteinizing hormone (pLH) in lieu of gonadotropin-releasing hormone (GnRH), for synchronizing ovulation, improved pregnancy rates (42 vs. 28%) in dairy cows, without increasing post-ovulation progesterone concentrations. Later, we found that a prolonged and elevated LH profile of pLH cows increased the expression of BMP-15, GDF-9, TGF-β1 proteins and genes associated with TGF-β superfamily pathway; these factors promote cumulus expansion and oocyte competence during preovulatory stage of the follicle. In the present study, we hypothesized that pLH exposure would alter the activation of the TGF-β pathway genes, which are involved in the development of preimplantation bovine embryos. Cyclic nonlactating Holstein cows (n = 16) were subjected to ovarian stimulation with 400 mg FSH and received either 100 μg GnRH or 25 mg pLH 24 h after the last FSH injection. Cows were artificially inseminated 12 and 24 h after GnRH or pLH injection. Serial blood samples were collected from before giving GnRH/pLH until 20 h after, for plasma LH analysis using an anti-bovine LH monoclonal antibody that cross-reacts equally with both bovine and porcine LH. Ova/embryos were recovered nonsurgically 6.5 d post-insemination. Total RNA was extracted from 3 to 4 morulae of each cow and qRT-PCR was performed with 3 biological replicates per treatment. Plasma LH concentrations and qRT-PCR data were analyzed using MIXED and GENMOD procedures of SAS, respectively. When LH concentrations (ng/mL) were compared in GnRH- and pLH-treated cows, mean LH was greater in GnRH- than pLH-cows (5.2 ± 0.2 vs.1.3 ± 0.2) from 30 min until 4 h post-treatment. However, mean plasma LH from 5 to 20 h post-treatment was higher (P < 0.001) in pLH- than in GnRH-treated cows (2.1 ± 0.1 vs. 0.6 ± 0.1). The relative abundance of PRS6KB-2, SMAD-2, and ACVR-1 in morulae, was about 2-, 3- and 3-fold higher, respectively, in cows subjected to pLH than GnRH treatment, but expression of THBS-4, BMP-2, BMPR1A and INHBA genes did not differ. Results indicate that a prolonged, higher-than-basal LH profile in pLH-treated cows increased the TGF-β signaling cascade and its components in bovine embryo, which may explain improved pregnancy rate following pLH-induced ovulation reported previously.