Salidroside, an active constituent from the root of Rhodiola rosea L., has multiple pharmacological effects, such as anti-cancer, anti-inflammatory and anti-oxidative properties, etc. However, its protective effect on bone tissue via regulating calcium homeostasis is yet to be determined. This study was performed to investigate if salidroside could protect against bone injuries induced by estrogen deficiency or hyperglycemia through modulating calcium homeostasis. Ovariectomized (OVX) mice and diabetic mice were treated with salidroside (20 mg/kg) for 6 weeks. Safranin O staining and micro-CT were performed on the distal metaphysis of femur. The calcium content in serum, urine and femur was measured, and the mRNA and protein expressions of regulators in kidney were determined by PCR and immunoblotting, respectively. Treatment with salidroside increased bone calcium level and decreased urinary calcium excretion, consequently attenuating the deteriorations of trabecular bone in both OVX mice and diabetic mice. 25-Hydroxyvitamin D-24 hydroxylase expression was down-regulated and vitamin D receptor expression was up-regulated in kidney of both OVX mice and diabetic mice upon to salidroside treatment, which also inhibited the ovariectomy-induced decrease in expression of renal transcellular calcium transporters and the diabetes-induced enhancement in renal calcium-sensing receptor (CaSR) expression. Taken together, salidroside exerted osteoprotective effects by improving calcium homeostasis via regulating vitamin D metabolism and transcellular calcium transporters as well as modulating CaSR expression in kidney.