Keratan sulfate (KS) is a sulfated glycosaminoglycan and has been shown to bind to sonic hedgehog (Shh), which act as a morphogen to regulate the embryonic spinal cord development. We found highly sulfated KS was present in the floor plate (including lateral floor plate) and the notochord . This expression colocalized with Shh expression. To understand the roles of KS, we analyzed the embryonic spinal cord of GlcNAc6ST-1, KS chain synthesizing enzyme, knock-out (KO) mice. At E12.5, the pMN domain, whose formation is controlled by Shh signaling, became shifted ventrally in GlcNAc6ST-1 KO mice. In addition, the expression patterns of Patched1 and Gli1, two Shh signaling reporter genes, differed between wild type (WT) and GlcNAc6ST-1 KO mice at E12.5. Next, we focused on cell types generated from the pMN domain; namely, motor neurons and subsequently oligodendrocytes. The number of PDGFR alpha(+) [a marker for oligodendrocyte precursor cells (OPCs)] cells was low in the E12.5 mutant spinal cord, while motor neuron production was increased. Thus the switch from motor neuron generation to OPC generation was delayed in the pMN domain. Furthermore, we investigated the cause for this delayed switch in the pMN domain. The number of Olig2, Nkx2.2 double-positive cells was less in GlcNAc6ST-1 KO mice than in WT mice. In contrast, the number of Olig2, Neurogenin2 (Ngn2) double-positive cells related to the motor neuron specification was significantly greater in the KO mice. These results indicate that KS is important for the late phase Shh signaling and contributes to motor neuron to OPC generation switch.