We investigated the role of glucosamine (GlcN) on the integrin β4/plectin complex and its role in the regulation of mouse embryonic stem cell (mESC) migration and proliferation. GlcN significantly decreased integrin β4 mRNA/protein expression, whereas plectin protein expression did not change. Also, decrease of integrin β4 expression caused reduction of integrin β4/plectin complex formation, and then increased cell migration. GlcN increased intracellular calcium influx and protein kinase C (PKC) phosphorylation followed by integrin β4 serine phosphorylation and reduction of the integrin β4/plectin complex. GlcN entered into the cell through glucose transporter 1 and then increased O-GlcNAc transferase (OGT) and the level of glycosylation (CTD110.6). Inhibition of OGT (OGT inhibitor; ST045849) increased integrin β4/plectin complex opposite with decreased cell migration. Moreover, GlcN increased O-GlcNAc-specificity protein 1 (Sp1) and nuclear translocated p-Sp1 stimulated calmodulin (CaM) expression, which combined with plectin. In addition, GlcN increased Akt glycosylation and glycogen synthase kinase-3β (GSK-3β) phosphorylation, and then Snail1 glycosylation. Snail small interfering ribonucleic acid (siRNA) reversed the reduction of integrin β4/plectin complex and dissociation of cell junctions (tight and adherent junction). GlcN increased cell migration, cell cycle regulatory proteins [cyclinD1, cyclin-dependent kinase 4 (CDK4), cyclinE, and CDK2], and the percentage of S phase cells, which were inhibited by a PKC inhibitor, CaM siRNA, or Snail1 siRNA. Additionally, GlcN maintained the undifferentiation status of ESCs. In conclusion, GlcN contributed to migration and proliferation of mESCs through integrin β4/plectin complex reduction via Ca²⁺/PKC, as well as the Sp1/CaM and Akt/GSK-3β/Snail1 signaling pathway.