Introduction: Fabaceae are a rich source of protease inhibitors. Methods: A proteinaceous protease inhibitor of 25 KDa designated as Macrotyloma uniflorum protease inhibitor (MUPI) was isolated from seeds of Macrotyloma uniflorum and purified to homogeneity by ammonium sulphate precipitation, DEAE Sepharose column and CNBr activated Sepharose 4B Trypsin affinity chromatography. The purity was checked by reverse phase HPLC. Long-term type 2 diabetes can lead to various biological complications, such as hypertension and heart-related diseases. The glucose uptake studies were carried out with the purified MUPI and it reveals its potential to be explored as a potent anti-hyperglycemic agent. MUPI was rendered safer on HepG2 cells after MTT cytotoxicity assay. Results: The results of glucose uptake studies suggest MUPI to be equally efficient to that of the positive control drug Metformin against diabetes mellitus. Conclusion: The significance of plant-based inhibitors for modulating carbohydrate breakdown and control of glycemic index is explored to reduce the risk factors and side effects of the available drugs. © 2018 Bentham Science Publishers.