Flavonoids are the active components of Herba epimedii (HEP), a commonly used herb for the management of osteoporosis in China over the centuries. The present study aims to characterise the in vivo effects of its total flavonoid (TF) fraction on bone properties and mineral metabolism as well as to study the mechanism involved in achieving its protective effects against ovariectomy (OVX)-induced bone loss. TF suppressed OVX-induced increase in urinary Ca excretion as well as loss of bone mass and strength at the distal femur in mice in a dose-dependent manner. The changes in urinary Ca excretion were inversely correlated with the expressions of renal Ca transport protein (CaBP-28K) and vitamin D receptor mRNA in OVX mice. TF (100 μg/g) treatment prevented the deterioration of trabecular bone microarchitecture induced by OVX in mice. In addition, TF treatment increased the expression of type I collagen and osteocalcin mRNA and the ratio of osteoprotegerin/receptor activator of NF-κB ligand mRNA, and suppressed the increase in IL-6 mRNA induced by OVX in the femur of mice. The present results indicate that the optimal dosage of the TF fraction of HEP for the improvement of bone properties and mineral metabolism in OVX mice was between 50 and 100 μg/g. Mechanistic studies indicated that TF might increase renal Ca reabsorption, stimulate the process of osteoblast formation as well as suppress the process of osteoclastogenesis in OVX mice.