We aimed to study the relationship between glucosamine and FoxO1/Notch in gluconeogenesis and maintenance of mouse embryonic stem cell (mESC) self-renewal. Glucosamine (GlcN) increased glucose production and gluconeogenic enzyme (G6Pase and PEPCK) expression. GlcN also increased the percentage of cells in S phase, number of cells, and the protein expression of cell cycle regulatory proteins that were blocked by 3-mercaptopicolinic acid (gluconeogenesis inhibitor) or glucose transporter (GLUT) 1 neutralizing antibody. GlcN increased the O-GlcNAc transferase (OGT)–dependent protein O-GlcNAc level. Moreover, inhibition of OGT (by ST045849) decreased glucose production. GlcN enhanced the expression of OGT-dependent O-GlcNAcylated Notch1 and then increased the translocation of cleaved Notch1 to the nucleus. Moreover, GlcN stimulated the translocation of O-GlcNAcylated FoxO1 to the nucleus. GlcN increased the binding between cleaved Notch1 and FoxO1 with CSL, a transcription factor, which was blocked by L-685,458 (γ-secretase inhibitor) or ST045849, respectively. Simultaneous blockage of cleaved Notch1 and FoxO1 also decreased the expression of G6Pase and PEPCK more significantly than that by inhibition of cleaved Notch1 alone or FoxO1 alone. In addition, GlcN maintained the undifferentiation status while depletion of Notch1 and FoxO1 for 3 days decreased Oct4 and SSEA-1 expression and alkaline phosphatase activity or increased the mRNA expression of GATA4, Tbx5, Cdx2, and Fgf5. In conclusion, GlcN-induced OGT activation mediated glucose production through cleaved Notch1 and FoxO1, which contributed to the regulation of maintenance of self-renewal in mESCs.