Simple SummaryThe accessory sex glands are responsible for producing seminal plasma, and thus play a vital role in reproduction and fertility. While the horse rarely suffers from some of the accessory sex gland diseases affecting other domesticated animals, glandular secretions can nonetheless affect semen quality and survival, and little is known about the effects of steroid hormones on glandular development and regulation. This study assessed the expression level and distribution of the steroid receptors AR, ESR1, and ESR2, and the steroidogenic enzymes 3ΒHSD, CYP17, and CYP19 in the equine accessory sex glands at various stages of life and demonstrated that sex steroid receptors are present in all equine glands throughout life. In contrast, steroidogenic enzymes were only weakly and variably expressed, suggesting that the accessory sex glands are not significant sites of steroidogenesis.The expression pattern and distribution of sex steroid receptors and steroidogenic enzymes during development of the equine accessory sex glands has not previously been described. We hypothesized that equine steroidogenic enzyme and sex steroid receptor expression is dependent on reproductive status. Accessory sex glands were harvested from mature stallions, pre-pubertal colts, geldings, and fetuses. Expression of mRNA for estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), androgen receptor (AR), 3β-Hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD), P450,17α hydroxylase, 17–20 lyase (CYP17), and aromatase (CYP19) were quantified by RT-PCR, and protein localization of AR, ER-α, ER-β, and 3βHSD were investigated by immunohistochemistry. Expression of AR, ESR2, CYP17, or CYP19 in the ampulla was not different across reproductive statuses (p > 0.1), while expression of ESR1 was higher in the ampulla of geldings and fetuses than those of stallions or colts (p < 0.05). AR, ESR1 and ESR2 expression were decreased in stallion vesicular glands compared to the fetus or gelding, while AR, ESR1, and CYP17 expression were decreased in the bulbourethral glands compared to other glands. ESR1 expression was increased in the prostate compared to the bulbourethral glands, and no differences were seen with CYP19 or 3β-HSD. In conclusion, sex steroid receptors are expressed in all equine male accessory sex glands in all stages of life, while the steroidogenic enzymes were weakly and variably expressed.