Oxyberberine (OBB), an important in vivo metabolite of berberine, exerts superior hypoglycemia effect. However, the underlying mechanism remains obscure. Heme oxygenase-1 (HO-1) holds a crucial status in the pathogenesis of diabetes. Previous research has indicated that OBB can specifically bind to hemoglobin and significantly up-regulated the HO-1 expression in diabetic rat. Based on cellular protection features of HO-1, this work aimed to probe the anti-diabetic effect of OBB and the association with the potential induction of HO-1 expression.A type 2 diabetic mellitus rat model was established. Glucolipid metabolism and insulin sensitivity were analyzed. Immunohistochemistry, Western blotting and in silico simulations were also performed.Administration of OBB or HO-1 inducer hemin significantly reduced fasting blood glucose level, blood fat, and inflammatory cytokine levels, while increased antioxidant capacity of pancreas. Meanwhile, OBB treatment remarkably stimulated liver glycogenesis and inhibited gluconeogenesis. Besides, OBB improved the glucose utilizing of muscle. Noteworthily, OBB inhibited the islet cell apoptosis and improved pancreatic function. In addition, OBB effectively improved the consumption of glucose in insulin-resistant HepG2 cells. Moreover, OBB also reduced oxidative stress, promoted glucose-elicited insulin secretion and enhanced expression of β-cell function proteins in INS-1 cells. Nevertheless, these effects were significantly reversed by treatment with Zincprotoporphrin (ZnPP). Additionally, in silico simulations indicated that OBB exhibited superior affinity with HO-1.OBB effectively ameliorated hyperglycemia, dyslipidemia, and insulin resistance, improved oral glucose tolerance, and maintained glucose metabolism homeostasis, at least in part, by promoting HO-1-mediated activation of phosphoinositide 3-kinase / protein kinase B (PI3K/Akt) and AMP-activated protein kinase (AMPK) pathways. These data eloquently suggest that OBB, as a novel HO-1 agonist, has good potential to be a promising candidate drug for the management of diabetes, and support a therapeutic role of HO-1 induction in diabetes that potentially paves the way to translational research.Copyright © 2022 Elsevier GmbH. All rights reserved.