Angels wing mushroom, Pleurocybella porrigens, is distributed worldwide in temperate areas. In 2004, seventeen people in Japan died of acute encephalopathy after eating this mushroom, most of whom were undergoing hemodialysis for chronic renal failure. Although the mechanism of the encephalopathy remains unclear, previous studies have reported vitamin D analogues, fatty acids, and saccharides as potential causative agents. Our group previously identified a lectin and six novel cytotoxic amino acids from the mushroom, all of which contain a β-hydroxyvaline unit attached to endogenous molecules, leading to the hypothesis that an aziridine amino acid (1, pleurocybellaziridine) with a carboxy group and geminal methyl groups on the aziridine ring is the common precursor. The instability of this molecule (prone to nucleophilic attack and ring opening by endogenous molecules) explains why it could not be isolated previously. To confirm its existence, we synthesized the proposed molecule and its esters (methyl and diphenylmethyl esters). The mushroom extract was esterified with CH₂N₂ or Ph₂CN₂, and the corresponding esters were isolated and purified. Their spectral data matched those of the synthetic compounds, confirming for the first time the presence of this labile amino acid in a natural source. The content of 1 in the mushroom was high (23 mg of diphenylmethyl ester from 4.0 g lyophilized fruiting bodies), and its absolute configuration was consistent with the synthetic counterpart. Toxicity assays showed that 1 significantly reduced the viability of rat CG4-16 oligodendrocyte cells at 10 μg/mL, while its methyl ester (11) exhibited weak toxicity, indicating that the carboxy group and aziridine skeleton are crucial for cytotoxicity. In summary, we synthesized this labile and toxic aziridine amino acid and elucidated its presence in P. porrigens.