In Agaricus bisporus, a common edible mushroom, we previously found several unidentified ninhydrin-positive compounds, with two identified as β-L-glutamylglycine and L-saccharopine. In this study, we characterized a unique glutamic acid conjugate (MX-O) from the mushroom. MX-O was isolated from the amino acid fraction of the 75% ethanol extract of the mushroom's fruit bodies using Dowex 50×4 column chromatography (eluted with 0.12 N HCl to obtain fraction F-II) followed by preparative paper chromatography. Acid hydrolysis of MX-O yielded L-glutamic acid and ethanolamine (identified via amino acid analyzer, DNP derivative TLC, and gas-liquid chromatography-mass spectrometry). The molar ratio of L-glutamic acid to ethanolamine was 1:1. Radioactivity incorporation experiments indicated glutamic acid was linked to ethanolamine through its β-carboxyl group, supported by electrophoretic mobility at pH 3.1 and insusceptibility to ninhydrin in the presence of Cu²⁺. MX-O exhibited chromatographic behaviors identical to authentic N-(β-L-glutamyl)ethanolamine, confirmed by co-chromatography. Thus, MX-O was identified as N-(β-L-glutamyl)ethanolamine—the first report of its occurrence in mushrooms. The concentration in fresh fruit bodies at stage 2 was approximately 0.5 μmol/g.