A number of recent studies have suggested that flavonols (a class of phytochemical with many biological activities), might exert protective effects against post-menopausal bone loss. In the present study, we compared naringenin (NG) and 8-prenylnaringenin (PNG), two major naturally occurring flavonols, on in vitro differentiation of osteoblasts and bone resorbing activity, of rat bone marrow stromal cells (BMSCs). Our results indicated that both compounds, at 10−6 m, enhanced BMSCs’ differentiation. Then effects of the two compounds at 10−6 m on ALP activity, osteocalcin secretion and calcium deposition, were compared over a time course. Numbers and areas of colonies stained for ALP (CFU-FALP) expression, and mineralized bone nodules, were histochemically analysed after 12 days and 16 days osteogenic induction, respectively. Expression of BMP-2, OPG, OSX, RUNX-2 genes and p38MAPK protein were examined using real-time PCR and western blotting, respectively. The data presented indicate that PNG, significantly enhanced the rat BMSCs’ differentiation and mineralization through the BMP-2/p38MAPK/Runx2/Osterix signal pathway, greater than did NG. In conclusion, PNG has a more pronounced ability to enhance osteoblast differentiation and mineralization, than NG.