Over the past 30 years, more than 260 apparently healthy villagers in Yunnan Province, southwest China, have suddenly died of an unknown cause (Yunnan Sudden Unexplained Death, SUD), which clusters in time and space during the rainy season and is epidemiologically linked to eating the mushroom Trogia venenata. This study isolated and characterized two unusual toxic amino acids, 2R-amino-4S-hydroxy-5-hexynoic acid (1) and 2R-amino-5-hexynoic acid (2), as well as the known toxin γ-guanidinobutyric acid (3) from T. venenata. Oral toxicity tests in mice showed LD50 values of 71 mg kg⁻¹ for 1 and 84 mg kg⁻¹ for 2. LC-MS/MS analysis of post mortem blood from a 27-year-old SUD victim revealed the presence of toxin 1, directly confirming he had eaten the mushroom before death. Animal experiments demonstrated that toxins 1 and 2 induced a 1.1–1.6-fold increase in serum creatine kinase (CK) levels (indicating muscle toxicity) and profound hypoglycemia (median 0.66 mmol L⁻¹ glucose within 2 hours of exposure, likely causing ATP depletion and neural cell death). A campaign warning villagers against eating T. venenata reduced SUD reports to zero in affected areas in 2010 and 2011. These findings provide conclusive evidence that toxins from T. venenata are the cause of Yunnan SUD.