The effect of various levels of proteins, sulfates, and molecular weight (Mw) of a sulfated-glycoprotein (NF3) from a sea cucumber, Stichopus japonicus, on nitric oxide (NO) releasing capacity from RAW 264.7 cells was investigated. The NF3 derivatives had various amounts of proteins (4.8~11.2%) and sulfates (6.8~25.2%) as well as different Mw (640.3×103~109.2×103 g/mol). NF3 was able to stimulate RAW 264.7 cells to release NO with lower protein contents, indicating that the protein moiety was not an important factor to stimulate macrophages. On the other hand, the NO inducing capacity was significantly reduced with decreased levels of sulfates and Mw, implying that sulfates and Mw played a pivotal role in activating RAW 264.7 cells. It was not clear why sulfates and a certain range of Mw were essential for stimulating macrophages. It appeared that certain levels of sulfates and Mw of sulfated-glycoproteins were required to bind to the surface receptors on RAW 264.7 cells.