Three mammalian fringe proteins are implicated in controlling Notch activation by Delta/Serrate/Lag2 ligands during tissue boundary formation. It was proved recently that they are glycosyltransferases that initiate elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats in the extracellular domain of Notch molecules. Here we demonstrate the existence of functional diversity among the mammalian fringe proteins. Although both manic fringe (mFng) and lunatic fringe (lFng) decreased the binding of Jagged1 to Notch2 and not that of Delta1, the decrease by mFng was greater in degree than that by lFng. We also found that both fringe proteins reduced Jagged1-triggered Notch2 signaling, whereas neither affected Delta1-triggered Notch2 signaling. However, the decrease in Jagged1-triggered Notch2 signaling by mFng was again greater than that by lFng. Furthermore, we observed that each fringe protein acted on a different site of the extracellular region of Notch2. Taking these findings together, we propose that the difference in modulatory function of multiple fringe proteins may result from the distinct amino acid sequence specificity targeted by these glycosyltransferases.