This study aimed to shed light on the protective and therapeutic anti-osteoporotic effects and mechanisms of action of grapefruit juice (GFJ) on prednisolone-induced osteoporosis a rat femoral fracture model. We found that treating rats with GFJ before and/or after prednisolone-induced osteoporosis resulted in increased bone density, total mineral content, and calcium content to counteract the osteoporotic effects of prednisolone. In parallel, the histological and ultrastructural results of the GFJ-treated groups correlated well with enhanced breaking strength of femurs subjected to a constant load. Furthermore, GFJ treatment before and after prednisolone-induced osteoporosis decreased plasma alkaline phosphatase and tartrate-resistant acid phosphatase activities and increased the level of insulin-like growth factor 1. Mechanistically, our immunohistochemistry study showed that GFJ ameliorated prednisolone-induced osteocalcin depletion, decreased receptor activator of nuclear factor kappa-B