Regulation of proteins by O-GlcNAc modification is becoming a major area of research. This reversible modification depends on glucose concentrations and, therefore, constitutes a powerful mechanism to regulate protein activities according to glucose availability. Its importance in glucose-dependent gene transcription is underlined by its role in pancreatic insulin biosynthesis (through PDX-1 and NeuroD1 O-GlcNAc modifications) and leptin synthesis in adipose tissue (through Sp1 O-GlcNAc modification). Moreover, in chronic hyperglycaemia, O-GlcNAc modifications of Sp1, p53 and NFκB participate in glucotoxicity, resulting in cardiovascular and renal alterations. The recent discovery by two independent groups that FoxO1 is regulated by O-GlcNAc modification provides a potential mechanism by which hyperglycaemia promotes gluconeogenesis and worsening of glucose intolerance, opening new research perspectives in the field.