Human sucrase enzyme is a key therapeutic target for type 2 diabetes. While sugarcane sucrase inhibitor (sucinh) modulates invertase activity thereby accumulates sucrose. Molecular level understanding of sucinh towards mammalian α-glucosidases is scarce. The interaction of sucinh with human sucrase was identified and the association of these proteins was confirmed using co-purification, co-immunoprecipitation and pull-down assay. In addition, microscale thermophoresis assay showed that sucinh has a tight binding with sucrase (Kd = 4.77 nM) and a better affinity over acarbose. Collectively, in vitro, ex vivo and in silico data revealed that sucinh is selective for intestinal sucrase. The M region (H5/6 loop) of sucinh identified at the protein-protein interface is shown to have high affinity over N and C regions. Whereas, the biolayer luminescent imaging and microscale thermophoresis on the synthetic peptide of 28-amino acids of M region has a weak dose-dependent binding with sucrase. However, the synthetic peptide did not show substantial inhibition of sucrase and amylase activities at low concentration. Naturally derived carbohydrate mimics were shown to have a positive impact at the in vitro conditions. The insights obtained in this study give clues towards a new class of bioactive therapeutic peptides for α-glucosidases. A new horizon towards polypeptides derived from food sources emerge as a promising strategy for dietary interventions for prediabetic conditions.,Copyright © 2018. Published by Elsevier B.V.