The present study was designed to investigate whether chondroitin sulfate (CS)-E, a CS structural isomer variant, alter the differentiation of macrophage cell line RAW264 cells to osteoclast-like cells. CS-B, CS-E, low molecular weight CS-E, synthetic chondroitin polysulfate (CPS) and heparin significantly inhibited the formation of tartrate-resistant acid phosphatase-positive multinuclear cells and pit formation on calcium phosphate (CaP)-coated plates. CS-E pre-coated on the CaP plate also inhibited pit formation. Digestion of CS on the cell surface by chondroitinase showed no effect on the osteoclastic differentiation of RAW264 cells whereas inhibitory effect on the differentiation of osteoblastic cell line MC3T3-E1. On the other hand, exogenously added fluoresceinlabeled CS-E directory bound to fibronectin and RAW264 cells. These results suggest that CS-E structure on the surface of osteoblasts or bone matrix binds to cell adhesion molecule such as integrin on the pre-osteoclastic cells and inhibits the differentiation into osteoclasts. CS-E may have a potential in treating bone defect if combined with CaP materials.