Reducing polysaccharide degradation through inhibition of α-amylases and α-glucosidases is one of the strategies for glycemic control in the treatment of diabetes mellitus. Here we report the identification and characterization of a new β-defensin-like miniprotein magnificamide-2 (Mgf-2), one of the most potent inhibitors of mammalian α-amylases with subnanomolar binding affinity (K of recombinant Mgf-2 is 0.029 nM against human pancreatic α-amylase, 0.057 nM against human salivary α-amylase, and 0.011 nM against porcine pancreatic α-amylase). Using nuclear magnetic resonance (NMR) spectroscopy, ensemble protein-protein docking, and molecular dynamics (MD) simulations, the key structural determinants responsible for this ultra-tight inhibition were identified, which was subsequently confirmed by site-directed mutagenesis experiments. The β1-β2 loop containing the inhibitory motif YIYH plays a crucial role in the inhibitor-enzyme interaction by forming an extensive hydrophobic interface with the hydrophobic "rim" around the α-amylase active site. The data obtained enhance our understanding of the molecular mechanisms underlying high-affinity α-amylase inhibition and provide valuable insights into the design of novel proteinaceous α-amylase inhibitors for the treatment and prevention of postprandial hyperglycemia.