Reduced L-glutathione (GSH) is a kokumi active tripeptide that enhances umami, salty, and sweet taste perceptions, probably via the calcium-sensing receptor (CaSR). In this study, we report that GSH is a partial agonist of the umami taste receptor (hTAS1R1/rTAS1R3). Using cellular assays, we revealed synergistic effects of GSH with L-glutamic acid (L-Glu) but not with 5′-ribonucleotides. Combining molecular modeling and mutagenesis studies, we mapped the GSH binding site located between the two lobes of the Venus Flytrap domain (VFT) of hTAS1R1. Interestingly, GSH is a weak agonist of the sweet taste receptor (hTAS1R2/hTAS1R3) and synergizes with sucralose via the rTAS1R3 subunit. Using the chimeric TAS1R3 receptor and site-directed mutagenesis, we showed that GSH binds to TAS1R3-VFT. This research provides increased understanding of the molecular interactions between GSH and TAS1Rs and suggests that the kokumi activity of GSH is more complex than affecting CaSR alone.