Background: Pergularia tomentosa L. is a milkweed tropical plant. In the Middle Est, the aerial parts of P. tomentosa are traditionally used as an anti-diabetic remedy. In order to find scientific evidence for the traditional use of this plant, the effect of an aqueous extract of P. tomentosa aerial parts by in vivo assay was investigated. Moreover, to achieve deeper insight into the chemical composition of the above-mentioned extract an analytical approach based on LC-ESI/LTQOrbitrap/MS/MSn was evaluated. Wistar rats were fasted overnights and diabetes mellitus induced using streptozotocin (50 mg/kg body weight). The rats were randomly and equally divided into four groups (n=5): group I (normoglycaemic control), group II (rats treated with streptozotocin 50 mg/kg body weight), group III (P. tomentosa extract 200 mg/kg body weight), group IV (normoglycaemic treated with P. tomentosa extract, 200 mg/kg body weight). At the end of the treatment period, they were anesthetized under diethylether. Vital organs (kidneys, liver and pancreas) and blood samples were obtained for histopathological, biochemical and haematological analysis.Results: P. tomentosa showed a reasonable reduction in blood glucose level. Probably, P. tomentosa effect on hyperglycemic and hyperlipidemic diabetic animals was associated to antioxidant properties, triglyceride levels, as well as the liver enzymes. Furthermore, the metabolite profile of the aqueous extract of P. tomentosa obtained by LC-ESI/LTQOrbitrap/MS/MSn highlighted the presence of double-linked cardenolides along with cardenolides and flavone glycosides. Conclusion: P. tomentosa decreased the glucose level and induced beneficial effect on lipid profile. This study confirms the capability of cardenolide glycosides to decrease the level of glucose in the blood.