In the investigation of chemical constituents in antiosteoporotic active fraction, 90% ethanol fraction of water extract of the rhizomes of Dioscorea spongiosa, twenty-two glycosides were isolated and their structures were elucidated. In order to know the role of each compound played in the fraction and to find the excellent antiosteoporotic constituent, they were subjected to some in vitro assay experiments with cell lines and an animal organ culture system: stimulation on osteoblasts proliferation and mineralization, inhibition on osteoclasts formation and inhibition on bone resorption. Glycosides 1, 3, 4, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19 and 21 showed significant stimulatory activity on proliferation of osteoblasts. Especially, pregnane glycoside 4, showed the strongest stimulatory activity. The stimulatory activity on osteoblasts mineralization of the compounds stimulating the proliferation significantly was examined and compounds 8, 10, 13 and 16 showed stronger stimulation on osteoblasts mineralization. Their inhibitory activity on bone resorption indicated that only glycosides 5, 13, 14 and 21 possessed significant inhibitory activity at high concentration. Glycosides 9, 11, 14, 18 and 19, isolated in large amount, all showed very strong inhibitory activity on formation of osteoclasts at high concentration, which may be induced by cytotoxicity to bone marrow cells and osteoblasts due to the observation under microscope. It implied that their inhibition on bone resorption may origin from their inhibition on formation of osteoclasts.