This study investigated non-protein amino acids in seeds and pods of Gymnocladus dioica. Ground seed meat and pods were extracted with 80% ethanol, defatted, and analyzed using AG 50W-X4 cation exchange resin column chromatography, preparative thin layer chromatography (TLC), high voltage electrophoresis (HVE), nuclear magnetic resonance (NMR), and mass spectrometry (MS). Previously reported non-protein amino acids including (2S,3R,4R)-β-hydroxy-γ-methylglutamic acid, (2S,3S,4R)-β-hydroxy-γ-methylglutamic acid, pipecolic acid, and 5-hydroxypipecolic acid were identified. Additionally, p-amino-phenylpropionic acid—essentially known as a synthetic compound—was purified from the seeds, representing its first natural occurrence. Non-protein amino acids in Gymnocladus seeds may function as toxins or feeding inhibitors to herbivores, and the discovery of p-amino-phenylpropionic acid in this species is significant as it had not been previously reported in nature.