Background: Diabetes mellitus (DM) is an endocrinological disorder characterized by increased blood glucose, which in turn leads to increased oxidative stress and development of other complicated disorders. The disorder is caused either by non-production of insulin by pancreatic -cells or development of insulin resistance. In recent years, diabetes is increasing rapidly. Although high blood glucose (hyperglycemia) is controllable through anti-diabetic drugs and/or insulin injection, rural people in Bangladesh prefer plant-based traditional medicines dispensed by traditional medicinal practitioners. Methods and findings: The present study objective was to conduct oral glucose tolerance test (OGTT) in glucose-challenged mice with methanol extract of Hibiscus sabdariffa calyces (MEHS); the calyces are widely used in Bangladesh for controlling hyperglycemia. At doses of 100, 200 and 400 mg per kg body weight, MEHS reduced blood glucose level by 55.8, 62.0 and 62.77%, respectively compared to control mice, which were untreated. Glibenclamide, by comparison, reduced blood glucose level by 61% at a dose of 10 mg per kg body weight. Furthermore, in silico molecular docking studies showed that a number of phytochemicals present in Hibiscus sabdariffa calyces have predicted low binding energies for human -amylase (PDB number: 2QV4) with the potential for inhibiting hyperglycemia. Conclusions: (MEHS) reduced blood glucose by nearly 63% at a dose of 400 mg/kg, which was comparable to that of glibenclamide at a dose of 10 mg/kg. In silico studies further indicated that a number of phytochemicals of the calyces of Hibiscus sabdariffa merits further evaluation towards new anti-diabetic drug discovery.