Excessively activated transforming growth factor-beta 1 (TGF-β1) exacerbates benign prostatic hyperplasia (BPH) by triggering epithelial-mesenchymal transition (EMT) as well as epithelial and stromal cell differentiation. Hesperidin (HSP), a flavanone rich in citrus peels, exhibits a safe anti-cancer activity with few side effects. Although HSP reportedly inhibits cell growth in prostate cancer, studies on BPH have not yet been reported. Thus, this study aimed to figure out the therapeutic effect of HSP and its underlying mechanisms in BPH models in vivo and in vitro. To evaluate the anti-BPH effect of HSP in vivo, rats were injected with testosterone propionate (TP; 10 mg/kg, s.c.), finasteride (5 mg/kg, p.o.), and HSP (50 and 100 mg/kg, i.p.) for four weeks. The in vitro efficacy of HSP was evaluated using two prostate cell models, BPH-1 and dihydrotestosterone-stimulated WPMY-1 cells, for studying the interaction between epithelial and stromal cells. Both in vivo and in vitro, HSP inhibited prostate cell proliferation by suppressing the expression of androgen receptor-related markers. In addition, HSP reduced the expression levels of inflammatory and mesenchymal markers by blocking TGF-β1 activation. Collectively, HSP alleviated BPH by attenuating prostate cell proliferation, the inflammatory response, and EMT by regulating the TGF-β1/Smad signaling pathway. Thus, these results provide evidence for a new therapeutic approach against BPH.