Nerve growth factor-β (NGF) is a seminal plasma protein that induces ovulation and has a luteotrophic effect in camelids, but little is known regarding the interactions of seminal plasma-derived NGF on the pre-ovulatory follicle in the bovine species. Our objectives were to assess the effects of purified bovine NGF on steroidogenesis and angiogenic markers in the bovine pre-ovulatory follicle. Two Holstein heifers were synchronized, and ovariectomy was performed via colpotomy when a pre-ovulatory follicle >12 mm was present. The pre-ovulatory follicle was excised from the ovary, fluid was aspirated, and the remaining tissue was dissected. The theca interna with adherent granulosa cells was peeled from the theca externa and surrounding stromal tissue and cut into small pieces (average weight: 5.3 ± 0.7 mg). The follicle tissue pieces (n = 24) were incubated in 0.5 mL of Eagle’s MEM culture medium supplemented with 1% l-glutamine, 1% nonessential amino acids, 1% penicillin-streptomycin, 1% insulin-transferrin-selenium, 10% fetal bovine serum, 40 ng/mL cortisol, 4 ng/mL LH, and 4 ng/mL FSH. Culture wells were either supplemented with 100 ng/mL NGF (n = 12) or left as an untreated control (n = 12). Medium was withdrawn and replaced with fresh medium at 3, 6, 12, 24, 48, and 72 h of culture and frozen at −80°C for hormone analysis and follicle tissue pieces were frozen to measure steroidogenic and angiogenic gene expression using qPCR. A general linear mixed model with repeated measures for hormone data and Kruskal-Wallis rank sum test for non-parametric data were performed. Treatment with NGF increased testosterone and (P < 0.01) and steroidogenic enzyme 17β-hydroxysteroid dehydrogenase (P = 0.04) in the follicle wall extracts. There was no effect of NGF on progesterone and estradiol (P ≥ 0.14) or other steroidogenic enzymes (P ≥ 0.31). Treatment with NGF reduced (P = 0.02) fibroblast growth factor 2 (Fgf-2) but did not alter other angiogenic factors (P ≥ 0.44). In conclusion, NGF affected steroidogenesis increasing testosterone production and reduced Fgf-2, a marker of cell remodeling.