Pumpkin seeds contain lignan phytoestrogens, which have the potential to retrieve menopausal syndromes caused by estrogen deficiency. This study aims to determine the estrogenic effects of the extract of pumpkin seeds (EPS). In vitro experiment was performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on CHO-K1 cells. In vivo experiments were conducted using 24 of 7-weeks-old female Sprague Dawley rats, divided into four treatment groups (ovariectomized [OVX], ovariectomized for treatment with 2-μM 17β-estradiol, ovariectomized for treatment with 500 mg/kg BW EPS, and ovariectomized for treatment with 1000 mg/kg body weight (BW) EPS) and two baseline groups (OVX and sham). Uterine weight, mammary glands, serum lipid, and bone density were observed after 30 days. In silico experiment was conducted through molecular docking of secoisolariciresinol and lariciresinol to the estrogen receptors (ERs). EPS exhibited a biphasic effect, inducing cell growth at 10-100 μg/mL and decreasing cell viability at 250-1000 μg/mL. EPS 1000 mg/kg BW improved uterine weight and retrieved mammary glands comparable with 17β-estradiol. Meanwhile, EPS 500 and 1000 mg/kg BW increased high-density lipoprotein (HDL), decreased low-density lipoprotein (LDL), and recovered bone density in correlation with the decreasing osteoclasts and increasing osteoblasts. The docking score of secoisolariciresinol with ERα and ERβ were −91.13 and −95.87, respectively, and lariciresinol were −80.91 and −90.29, respectively. These results demonstrated that EPS performs estrogenic properties in rats model and potential to overcome symptoms caused by estrogen deficiency. KEYWORDS estrogenic properties, estrogen receptors, ovariectomized rat, pumpkin seeds