Six amentoflavone-type biflavonoids, bilobetin (1), ginkgetin (2), 4',7''-di-O-methyl-amentoflavone (3), 7-O-methyl-isoginkgetin (4), sciadopitysin (5), and 7,4',7'',4'''-O-methyl-amentoflavone (6), were isolated from the EtOAc fraction of Cephalotaxus koreana Nakai (Cephalotaxaceae) by bioactivity-guided fractionation technique using primary cultures of mouse osteoblasts as an in vitro assay system. Among the six biflavonoids isolated, bilobetin (1), sciadopitysin (5), and 7,4',7'',4'''-O-methyl-amentoflavone (6) significantly increased osteoblast differentiation as assessed by alkaline phosphatase activity, collagen synthesis, and mineralization. Considering structure-activity relationship, methoxyl groups at 4' and 4''' in the B rings in amentoflavone-type biflavonoid might be important in osteoblast differentiation. Taken together, our present study suggests therapeutic potential of biflavonoids against bone diseases such as osteoporosis.