beta1,3-N-Acetylglucosaminyltransferase-7 (beta3Gn-T7) has been identified as an anti-migration factor for a lung cancer cell line but its enzymatic activity has not yet been characterized. Here we show that beta3Gn-T7 efficiently acts on keratan sulfate-related glycans including Galbeta1-->4(SO(3)(-)-->6)GlcNAcbeta1-->3Galbeta1-->4(SO(3)(-)-->6)GlcNAc (L2L2), while lacto-N-tetraose and lacto-N-neo-tetraose were poor substrates. Moreover, we found that among the other five beta3Gn-Ts and i antigen-producing beta3Gn-T (iGn-T), beta3Gn-T2 and iGn-T act well on L2L2, although these specific activities were lower than those for a tetraantennary N-glycan. These results indicate that beta3Gn-T7 is the one that most efficiently elongates L2L2 and may be involved in the biosynthesis of keratan sulfate.