Benign prostatic hyperplasia (BPH) is one of the most common diseases in the urinary system of elderly men. Pao extract is an herbal preparation of the bark of the Amazon rainforest tree Pao Pereira (Geissospermum vellosii), which was reported to inhibit prostate cancer cell proliferation. Herein we investigated the therapeutic potential of Pao extract against BPH development in a testosterone-induced BPH rat model. The administration of testosterone induced the prostate enlargement, compared with the sham operated group with vehicle treatment. The BPH/Pao group showed reduced prostate weight comparable with BPH/finasteride group. Notably, Pao treatment did not significantly reduce body weights and sperm number of rats, compared with the control group. Furthermore, Pao extract treatment reduced the proliferative index in prostate glands and testosterone-induced expression levels of AR, as well as androgen-associated proteins such as SRD5A1 and PSA. Moreover, Pao extract and its active component, flavopereirine, induced cytotoxicity on human prostate epithelial RWPE-1 cells in a dose- and time- dependent manner with G2/M arrest. Consistently, Pao extract and flavopereirine suppressed the expression levels of SRD5A1, AR and PSA, respectively. Together, these data demonstrated that Pao extract suppresses testosterone-induced BPH development through inhibiting AR activity and expression, and suggested that Pao extract may be a promising and relative safe agent for BPH.