Gurmarin is a polypeptide isolated from the Indian plant Gymnema sylvestre, which specifically suppresses sweet taste in rodents without affecting responses to other basic taste stimuli, such as HCl, NaCl, and quinine. Although the exact mechanism of gurmarin inhibition is not known, it has been shown that gurmarin acts via the T1R2/T1R3 sweet taste receptor. The gurmarin molecule is made of 35 amino-acid residues and three intramolecular disulfide bridges. We report herein the 1.45 Å X-ray structure of gurmarin heterologously produced using the yeast Pichia pastoris. The structure revealed a typical knottin fold, which is compared with previously reported NMR solution structures. The atomic structure at this resolution allowed us to highlight a flexible region involving hydrophobic amino acid residues previously identified as a putative binding motif for the rat sweet taste receptor. By combining cellular based receptor assay and site-directed mutagenesis of gurmarin, we revealed that several amino acid residues located in this hydrophobic cluster of gurmarin severely affect rat sweet taste receptor inhibition. This study demonstrates that gurmarin can be used as a worthwhile tool to decipher the mechanism of sweet taste inhibition.