Recent data support that diabetes might be a conformational disease. Certainly, hyperglycaemia causes a broad range of deleterious effects that might facilitate protein aggregation. We have evaluated the effects of hyperglycaemia on antithrombin, a conformationally sensitive serpin with a potent anticoagulant role. Moreover, these studies might also help to understand the thrombotic risk associated to diabetes. We incubated in vitro plasma and purified antithrombin and human hepatoma cells (HepG2) with methyl-glyoxal and glucose. Moreover, a mouse model of acute diabetes was generated with streptozotocin. Antigen, anti-FXa activity, heparin affinity and conformational features of antithrombin were analysed. Histological and intracellular features and distribution of antithrombin in HepG2 and livers of mice were also evaluated. Hyperglycaemia in vitro induced a transition of antithrombin to a form with low heparin affinity that explained the loss of anticoagulant activity, without generation of abnormal conformers (polymers or latent antithrombin). However, these effects were not observed on circulating antithrombin from diabetic mice. In contrast, hyperglycaemia in vivo had significant effects on intracellular antithrombin, which was retained, forming microaggregates within the lumen of dilated cisterns of the endoplasmic reticulum. These effects explained the moderate type I deficiency observed in diabetic mice. Similar intracellular consequences were observed for another hepatic serpin, alpha1-antitrypsin. Our data further support that diabetes has conformational effects on structurally sensitive proteins. These effects on antithrombin, the main natural anticoagulant, might contribute to the hypercoagulable status of diabetic patients.