Lactisole, an inhibitor of the human sweet taste receptor, has a 2-phenoxypropionic acid skeleton and has been shown to interact with the transmembrane domain of the T1R3 subunit (T1R3-TMD) of the receptor. Another inhibitor, 2,4-DP, which shares the same molecular skeleton as lactisole, was confirmed to be approximately 10-fold more potent in its inhibitory activity than lactisole; however the structural basis of their inhibitory mechanisms against the receptor remains to be elucidated. Crystal structures of the TMD of metabotropic glutamate receptors, which along with T1Rs are categorized as class C G-protein coupled receptors, have recently been reported and made it possible to create an accurate structural model for T1R3-TMD. In this study, the detailed structural mechanism underlying sweet taste inhibition was characterized by comparing the action of lactisole on T1R3-TMD with that of 2,4-DP. We first performed a series of experiments using cultured cells expressing the sweet taste receptor with mutations and examined the interactions with these inhibitors. Based on the results, we next performed docking simulations and then applied molecular dynamics-based energy minimization. Our analyses clearly revealed that the (S)-isomers of both lactisole and 2,4-DP, interacted with the same seven residues in T1R3-TMD and that the inhibitory potencies of those inhibitors were mainly due to stabilizing interactions mediated via their carboxyl groups in the vertical dimension of the ligand pocket of T1R3-TMD. In addition, 2,4-DP engaged in a hydrophobic interaction mediated by its o-Cl group, and this interaction may be chiefly responsible for the higher inhibitory potency of 2,4-DP.