Prolactinomas are harmful to human health, and the clinical first-line treatment drug is bromocriptine. However, 20% prolactinomas patients did not respond to bromocriptine. Hordenine is an alkaloid separated from Fructus Hordei Germinatus, which showed significant antihyperprolactinemia activity in rats. The aim of this study was to explore the effect and mechanism of hordenine on prolactinomas in rats. The study used estradiol to induce prolactinomas, which caused the activation of the pituitary mitogen-activated protein kinase (MAPK) pathway in rats significantly. The treatment of hordenine restored estradiol, induced the overgrowth of pituitary gland, and reduced the prolactin (PRL) accumulation in the serum and pituitary gland of rats by blocking the MAPK (p38, ERK1/2, and JNK) activation and production of inflammatory cytokines, tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and interleukin-6 (IL-6). The antiprolactinoma effect of hordenine was mediated by inhibiting the MAPK signaling pathway activation in rats.