Abstract Osteoporosis is a disease of bones that leads to an increased risk of fracture. Fructus of Psoralea corylifolia L. (scurfpea fruit) is commonly utilized for treating bone fractures and joint diseases for thousands of years in China. This study was aimed to screen active principles, which might have the potency to stimulate osteoblasts proliferation and differentiation from scurfpea fruit. A HPLC method was established to analyze the main components in scurfpea fruit. Totally 11 compounds have been identified by comparing their retention time with correspondent standard substances. The MTT and ALP methods were utilized for the assay of osteoblasts proliferation and differentiation activity. Icariin, a prenylated flavonoid glycoside was treated as the positive control. Bavachin and isobavachin significantly stimulated cell proliferation, while bakuchiol exhibited stronger effect to enhance osteoblasts differentiation. All these compounds were found with a characterized structure that in each of their molecule backbones, a prenylated side chain was attached. These results lead to a hypothesis that prenyl group might be crucial to exhibit the activity. The structure–effect relationship of these compounds with prenyl group in mouse primary calvarial osteoblasts needs to be explored in further research.