Nerve Growth Factor-β (NGF) is a seminal plasma protein that regulates sperm physiology in bulls, yet its production and association with fertility remain unclear. The objective of this study was to describe NGF distribution in bull accessory sex glands, quantify its seminal plasma concentrations, and determine its association with sire conception rates. Accessory sex glands were collected post-mortem from mature bulls (n = 3). Quantitative PCR and immunohistochemistry were performed in tissue samples. Pre-ejaculate and sperm-rich fraction of seminal plasma from Angus (n = 42) and sperm-rich fraction from Holstein (n = 10) bulls were collected by electroejaculation. Holstein (n = 76) and Jersey (n = 14) bulls were collected by artificial vagina, and those with available sire conception rate scores were allocated to: (1) negative/0 (n = 15); or (2) positive (n = 45). Seminal plasma NGF concentrations were measured using ELISA. Relative abundance of NGF mRNA was greatest in the vesicular, intermediate in ampulla, and lowest in prostate and bulbourethral glands (P ≤ 0.03). The most intense NGF staining was detected in the ampulla and vesicular glands (P < 0.05). Concentrations of NGF were greater in the sperm-rich fraction than in the pre-ejaculate and in samples collected by artificial vagina (P < 0.01). Seminal plasma NGF concentrations were greater in bulls with positive sire conception rate than those with negative/0 (P = 0.05). This study determined that NGF is produced in the ampulla and vesicular glands of bulls and is secreted into the sperm-rich fraction of the ejaculate. The positive association of NGF with bull fertility warrants further investigation.