Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, ss1,3-N-acetylglucosaminyltansferase-7 (ss3GnT7) and ss1,4-galactosyltransferase-4 (ss4GalT4), in production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than 4 repeats of GlcNAc-sulfated N-acetyllactosamine unit in presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of ss3GnT7/ ss4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of ss3GnT7 or ss4GalT4 was suppressed by siRNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.