To investigate the effects of 8-prenylnaringenin on osteoblasts in vitro under the conditions of the ability to differentiate and biomineralization. Neonatal SD rat skull was segregated, bone cells were obtained by enzyme digestion and cultured in MEM containing 10% FBS. Three days later the culture medium was changed at the first time, serial subcultivation was proceeded until cells covered with 90% culture dish. The 8-prenylnaringenin final concentration is 1×10-6 mol/L. Under the induce condition, the Alkaline phosphatase activity, osteocalcin and calcium salt sediment yield were measured at the 3rd, 6th, 9th, 12th day. At the 12th day, histochemistry dyeing was carried for calcified tubercle and ALP. Total RNA was isolated and the gene expression of bFGF, IGF-1, osterix, BMP-2 and Runx-2 were investgated by RT-real time PCR. 8-Prenylnaringenin significantly advanced osteoblasts to differentiate and biomineralization. Raised the ALP activity and calcium salt sediment yield and osteocalcin and COL-Ⅰ, increased calcified tubercle amount were manifested. Besides, 8-Prenylnaringenin also can enhance the mRNA level of bFGF, IGF-1, osterix, BMP-2 and Runx-2. The 8-prenylnaringenin with final concentration 1×10^-6 mol/L can predominantly promote ROB differentiation and biomineralization.