Background: Osteoporosis is a multifactorial bone disease that progresses without notice until attaining a chronic stage. It causes socio-economic burden, higher mortality rate, and increased bone fractures. Ononin, an isoflavone present in numerous herbal Chinese medicinal formulations such as Astragali radix and red clover, has been reported to have potent therapeutic properties. Objectives: In this study, we aimed to evaluate the efficacy of ononin against ovariectomy(OVX)-induced osteoporosis in female rats. Materials and Methods: Ovaries were removed surgically to develop the animal model of OVX. Animals (n = 4) were grouped into control, OVX induced, OVX rats treated with ononin (10 mg/kg body weight), and OVX rats treated with 20 mg/kg body weight of ononin. Results: OVX rats treated with ononin showed improved body weight, uterus index, inflammatory and bone markers, serum lipid profile, and calcium level. Ononin downregulated the expression of estrogen (E2), acid phosphatase, and bone gamma-carboxyglutamate (Gla) protein in OVX rats. It also restored trabecular area and thickness, which was evidenced by histomorphometrical and histopathological examination. Ononin attenuated the expression of alkaline phosphatase, osterix, and Runx2 in OVX rats. Conclusion: In summary, our results suggest that ononin could be used as an ideal therapeutic agent to prevent and treat bone-associated disorders.