Angiogenesis impairment in hyperglycemic patients represents a leading cause of severe vascular complications of both type-1 and -2 diabetes mellitus (DM). Angiogenesis dysfunction in DM is related to glycemic control, however molecular mechanisms involved are still unclear. Fibroblast Growth Factor-2 (FGF-2) is a potent angiogenic factor and, according to previous evidence, may represent a key target of molecular modifications triggered by high sugar exposure. Therefore, purpose of this study was to investigate whether short incubation with hyperglycemic levels of glucose affected FGF-2 and whether glucose-modified FGF-2 was detectable in vivo. Biochemical analyses carried out with SDS-PAGE, fluorescence emission, mass-spectrometry, immunoblot and competitive ELISA experiments, demonstrated that human FGF-2 undergoes a rapid and specific glycation upon 12.5-50 mM glucose exposure. In addition, FGF-2 exposed for 30min to 12.5 mM glucose lost mitogenic and chemotactic activity in time- and dose-dependent way. Under similar conditions, binding-affinity to FGFR1 receptor was dramatically reduced by 20-fold, as well as FGFR1 and Erk-1/2 phosphorylation, and FGF-2 lost about 45% of angiogenic activity in two different in vivo angiogenic (Matrigel and Chorio-Allantoic-Membrane) assays. Such glucose-induced modification was specific, since other angiogenic growth factors, namely platelet derived growth factor (PDGF-BB) and placental derived growth factor (PlGF) were not significantly or markedly less modified. Finally, for the first time glycated-FGF-2 was detected in vivo, in tissues from hyperglycemic non-obese diabetic (NOD) mice, in amount significantly higher than in normoglycemic mice. In conclusion, hyperglycemic levels of glucose may strongly affect FGF-2 structure and impair its angiogenic features and endogenous glycated-FGF-2 is present in diabetic mice, indicating a novel pathogenetic mechanism underlying angiogenesis defects in DM.