Arginine vasopressin (AVP) is a neuropeptide involved in the regulation of fluid balance, stress, circadian rhythms, and social behaviors. In the brain, AVP is tightly regulated by gonadal steroid hormones in discrete regions with gonadectomy abolishing and testosterone replacement restoring normal AVP expression in adult males. Previous studies demonstrated that 17β-estradiol, a primary metabolite of testosterone, is responsible for restoring most of the AVP expression in the brain after castration. However, 5α-dihydrotestosterone (DHT) has also been shown to play a role in the regulation of AVP expression, thus implicating the involvement of both androgen and estrogen receptors (ER). Furthermore, DHT, through its conversion to 5α-androstane-3β,17β-diol, has been shown to modulate estrogen response element-mediated promoter activity through an ER pathway. The present study addressed two central hypotheses: 1) that androgens directly modulate AVP promoter activity and 2) the effect is mediated by an estrogen or androgen receptor pathway. To that end, we overexpressed androgen receptor, ERβ, and ERβ splice variants in a neuronal cell line and measured AVP promoter activity using a firefly luciferase reporter assay. Our results demonstrate that DHT and its metabolite 5α-androstane-3β,17β-diol stimulate AVP promoter activity through ERβ in a neuronal cell line.