Ethnopharmacological relevance: Aristolochia triangularis Cham. has been used in Brazilian traditional medicine for various therapeutic purposes, including as a leaf-based infusion for diabetes management. Aim of the study: This study was designed to chemically characterize an infusion of in natura A. triangularis leaves and evaluate the in vivo anti-hyperglycemic properties of this infusion. Materials and methods: Chemical composition was examined using liquid-liquid extraction procedure, chromatographic methods, NMR, and LC-MS/MS. The in vivo antihyperglycemic activity of the freeze-dried infusion of A. triangularis leaves (Inf-L-At) was assessed using oral glucose tolerance test (OGTT). Initially, normoglycemic male rats were pre-treated with orally administered InfL-At at doses of 62.5, 125, and 250 mg/kg for two consecutive days. On the day of the OGTT, fasting animals received a glucose load (4 g/kg) 30 min after treatment with Inf-L-At, and the blood glucose levels were verified at 15, 30, 60, and 180 min. Intestinal maltase, lactase, and sucrase activities and muscle and liver glycogen contents were also assessed after the OGTT. Results: Inf-L-At extract led to glycemic reduction with no doseresponse at 15, 30, and 60 min comparable to that of the antidiabetic drug glibenclamide and was accompanied by an increase in hepatic and muscle glycogen contents. Additionally, there was a significant statistically decrease in the in vitro activity of disaccharidases. Maltase and sucrase activities were inhibited at all doses, whereas lactase activity was inhibited only at 62.5 and 250 mg/kg. In total, 75 compounds were found in the infusion, including seven new ones, (7S*,8S*,7'S*,8'R*)-4,4'-dihydroxy-3,3'-dimethoxy-7,9'-epoxylignan-7'-ol; 4'-hydroxy-3'-methoxy-3,4-methylenedioxy-7,9'-epoxylignan-9,7'-diol; triangularisines A, B, and C; N-ethyl-N- methyl-affineine; and N-methyl pachyconfine, and one previously not described as a natural product, epi- secoisolariciresinol monomethyl ether. Conclusion: The results demonstrated the anti-hyperglycemic activity of the infusion from A. triangularis leaves and showed that it is a rich source of lignoids, alkaloids, and glycosylated flavonoids, which are known to exhibit antidiabetic effects and other biological properties that can be beneficial for patients with chronic hyperglycemia, thus certifying the popular use of this herbal drink.