Nerve growth factor-β (NGF) is a seminal plasma protein that stimulates bovine theca cell proliferation and steroidogenesis in vitro and is luteotrophic in vivo, yet there is little is known on how it alters ovulation and corpus luteum formation in cattle. Our objectives were to assess the effects of systemic purified bovine NGF on vascularity and steroidogenesis of the pre-ovulatory follicle and subsequent corpus luteum (CL) in cattle. Holstein heifers (n = 12) were synchronized using a 5-d CIDR-Synch and randomly allocated to 1 of 2 treatment groups: (1) CONT, 12 mL of PBS; or (2) NGF, 250 µg purified NGF in 12 mL of PBS administered intramuscularly at time 0 (presence of pre-ovulatory follicle). A second replicate was performed using a crossover ~1 mo after the first replicate. Transrectal ultrasonography with Doppler and blood sampling were performed every 4 h from 0 to 32 h to evaluate follicle size and vascularity, ovulation time, and estradiol concentrations. Ultrasonography was then performed daily to assess CL size and vascularity, and blood was obtained every 2 d to measure progesterone concentrations. On d 9 and 14, a CL biopsy was performed to assess mRNA expression of steroidogenic enzymes and LH receptor and the histological ratio of small to large luteal cells. Statistical analyses were performed using a general linear mixed model with repeated measures using R Version 3.4.3. Treatment with increased follicle diameter (P = 0.02) but did not alter estradiol (P = 0.95) or ovulation time (P = 0.42). Treatment with NGF tended to increase CL diameter (P = 0.10) and increased progesterone concentrations (P = 0.04). There was a higher percentage of small luteal cells (P < 0.01) and a tendency for increased LH receptor gene expression (P = 0.09) in NGF-treated heifers. Steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase were increased (P ≤ 0.05) in NGF-treated heifers. Treatment with NGF did not alter the vascularity of the follicle or CL (P ≥ 0.16). Purified NGF interacted with the pre-ovulatory follicle and altered downstream CL formation and function.