Osteoporosis increases bone fragility and fractures. Preptin hormone is regulated by moderate exercise training and increases bone formation. Therefore, this study was conducted to see how estradiol administration and moderate exercise training affected osteoporotic changes in ovariectomized (OVX) rats. To achieve this aim, 36 healthy adult female Wistar albino rats were randomized into Sham, OVX, ovariectomized estradiol-treated (OVX + E) (OVX + E rats were treated using subcutaneous estradiol benzoate 2.5 μg/kg body weight/day), ovariectomized practicing moderate exercise training, ovariectomized estradiol-treated and practiced a moderate exercise training, and ovariectomized alendronate-treated (OVX + Alen) (OVX + Alen rats were treated orally with alendronate 3 mg/kg body weight/week) groups. Alendronate was used as a standard anti-osteoporotic drug. Moderate exercise training, including therapy with estradiol and alendronate for OVX rats began on the fourth week and lasted for six weeks. Results showed that OVX rats had estrogen and preptin deficiency in serum. These deficiencies were associated with a significant increase in bone resorption biomarkers (urinary deoxypyridinoline and hydroxyproline), and bone formation biomarkers (serum osteocalcin and bone-specific alkaline phosphatase). Also, serum pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin-6) were increased, while bone osteopontin (OPN) expression was decreased. Subsequently, the osteoporotic alterations were verified based on histopathological changes. From the results, estradiol therapy and moderate exercise training significantly improved these findings to the same extent as that of the standard alendronate treatment. Therefore, through their anti-inflammatory properties, increasing bone OPN expression, and regulating serum preptin; estradiol therapy and moderate exercise training can reduce osteoporotic alterations in OVX rats. Thus, combined estradiol therapy and moderate exercise training could be a promising potential therapeutic protocol to reduce postmenopausal osteoporosis. Also, targeting serum preptin and bone osteopontin regulation could have a critical role in the treatment of postmenopausal osteoporosis.