Many parts of the plant Linn. are widely investigated for their biological properties. Purified Arbortristosides from seeds are reported as anticancer, anti-leishmania, anti-inflammatory, anti-allergic, immunomodulatory and antiviral. The present study elaborates on the isolation, structural and functional characterization of Arbortristoside-C and its inhibition properties against alpha-glucosidase, an important target for diabetes mellitus. Arbortristoside-C is purified from seeds of by extraction using polar fractionation and chromatographic techniques. Arbortristoside-C has been characterized using Ultra Violet (UV), Mass (MS), Infra-Red (IR) and Nuclear Magnetic Resonance (NMR). Inhibition kinetics and Isothermal Titration Calorimetry (ITC) were used for activity and binding characteristics of acarbose and Arbortristoside-C using in-house purified α-glucosidase from . Modeling, docking and structural comparison with acarbose bound structure revealing the similar binding characteristics of Arbortristoside-C which include interaction with catalytic acid/base Aspartic acid residue. Cytotoxicity assay revealed that 100 µg/ml is the maximum toxic-free concentration of Arbortristoside-C. The purified Arbortristoside-C showed inhibition against mammalian α-glucosidase, suggesting its potential to treat Diabetes mellitus.