Radix Paeoniae Alba (RPA) is widely used in clinical treatment for gynecological diseases, particularly abnormal menstruation, menstrual pain, and breast tenderness; however, no scientific evidence base links RPA to estrogen replacement therapy. In this study, we characterize estrogenic activity of RPA using immature and ovariectomized (OVX) mice together with in vitro studies focus on estrogen receptor (ER) pathway for molecular mechanism. RPA treatments demonstrated significant estrogenic activity, as indicated by promoting the development of uterus and vagina in immature mice, reversing the atrophy of uterus and vagina in OVX mice, up-regulating the expressions of ERα and ERβ at protein and mRNA level in reproductive tissues. Meanwhile, RPA significantly increased serum estradiol and clearly decreased serum luteinizing hormone and follicle-stimulating hormone of immature/OVX mice. Moreover, RPA could induce ER positive MCF-7 cell from S-phase to G2 stage and induce proliferation and no influence on ER negative MDA-MB-231 cell. RPA could bind with ERα and ERβ and significantly stimulate ERα/β-estrogen response element (ERE) luciferase reporter gene expression. All activities were inhibited by the ER antagonist ICI 182,780. This study illustrates RPA exerts estrogenic effects by stimulating biosynthesis of estrogen in circulation, up-regulating ERs in target tissues, and mimicking the estrogen through ER-ERE-dependent pathway.