Epimedium brevicornu Maxim., one of the most frequently used traditional Chinese medicines for thousands of years, is prescribed as having "bone strengthening" function and the ability to cure bone diseases. The present study evaluated the osteogenic effects of anhydroicaritin (1) and 2"-hydroxy-3"-enanhydroicaritin (2) isolated from E. brevicornu by activity-guided assay. Treatment with 1 and 2 improved the proliferation of murine osteoblastic MC3T3-E1 cells at doses of 10(-7)-10(-5) mol/L and 10(-7)-10(-6) mol/L, respectively, in the 72-hour culture period. Enzyme linked immunosorbent assay and histochemical staining demonstrated that both of these two prenyl-flavonoids significantly promoted the differentiation of MC3T3-E1 cells by enhancing the level of ALP activity in the cells. Alizarin Red staining and mineralized nodule quantification showed that 1 and 2 had the potential of stimulating the formation of mineralization nodules and further speeding up the formation of bone, indicating that both compounds might be potential candidates for bone regenerative medicine.