The objectives of this study were to determine the effects of nerve growth factor-β (NGF), purified from bulls' seminal plasma and administered at the time of artificial insemination (AI), on progesterone post-AI, interferon-stimulated genes (ISG), and pregnancy per AI (P/AI) for lactating Holstein dairy cows enrolled in a timed-AI protocol. We hypothesized that administration of NGF at the time of AI would increase plasma progesterone post-AI, upregulate relative abundance of ISG, and improve P/AI in lactating dairy cows. Holstein cows (n = 557) from a single commercial dairy farm were blocked by parity and randomly assigned to receive an intramuscular injection containing 296 µg of bovine purified NGF at the time of AI, diluted in 2 mL of phosphate-buffered saline (NGF: n = 275), or receive only the 2 mL of phosphate-buffered saline (control: n = 282). Plasma progesterone and corpus luteum size were assessed in a subset of cows (NGF: n = 32; control: n = 36) at d 7, 14, and 19 post-AI. Relative mRNA abundance of ISG (ISG15, MX1, MX2, and RTP4) was assessed in peripheral blood leukocytes on d 19 post-AI. Pregnancy diagnosis was performed at 37 and 65 d post-AI. There was an interaction effect between treatment and parity for plasma progesterone; however, plasma progesterone and ISG did not differ between treatments. There were no effects of NGF for P/AI at 37 d post-AI (NGF = 40.0% vs. control = 41.6%), 65 d post-AI (NGF = 36.0% vs. control = 38.1%), and for pregnancy loss (NGF = 8.4% vs. control = 7.7%). The current study revealed that effects to NGF in lactating Holstein cows were minor and contingent with parity for progesterone, and no improvement in ISG relative abundance and P/AI were observed.