Background: Dillenia pentagyna Roxb. (Dilleniaceae) is an endangered medicinal plant, commonly known as “agai” and distributed randomly in the Terai belt of North East states of India. D. Pentagyna extract has been studied for therapeutic possessions. Objective: The proximate parameters and high-performance thin layer chromatography (HPTLC) analysis in the same plant of D. pentagyna of four-time harvest stage during the maturation of fruits were studied and also the antihypoglycemic activity of hydroalcoholic extract of D. pentagyna fruit. Materials and Methods: The proximate compositions were determined by measuring the amount of water removed from the food AOAC method and the chemical fingerprinting was carried out by HPTLC method. An acute oral toxicity study was carried out in healthy male Wister rats (110 g). The dose was finally made to 200 and 400 mg/kg body weight for oral administration after the LD50 estimation. Results: The most phenolic concentration was continuously decrease with during the maturation of fruits. After 21 days of treatment, level of blood glucose, as well as albumin, protein, creatinine, urea, and uric acid were significantly decreased when compared with the diabetic control. Hydroalcoholic (80% alcohol to 20% DW) extracts will be subjected to further extensive studies to isolate and identify their active constituents which are useful for against antidiabetic. Conclusion: D. pentagyna fruit could act as a source of functional compounds for the control of diabetes mellitus.