Gleditsia japonica Miquel (Leguminosae) is used in oriental traditional medicine as a diuretic and expectorant, and its seeds contain polyamines, phenols, alkaloids, flavonoids, carbohydrates, saponins, steroids, coumarins, and amino acids. In a continuing search for biologically active compounds from plants, a new purine alkaloid glucoside designated locustoside A was isolated from the methanol extract of G. japonica seeds. This report describes the purification and structure elucidation of locustoside A. The structure was determined as 7-b-D-glucopyranosyl-3-(3-methyl-2-butenyl)-isoguanine by interpretation of spectroscopic data (ESIMS, HRESITOFMS, IR, UV, NMR) and confirmed by X-ray crystallographic analysis. The absolute configuration of glucose was determined as D by GC-MS analysis of the trimethylsilylated thiazolidine derivative after acid hydrolysis. Locustoside A is a 3-prenylated purine alkaloid glucoside, a rare type of compound since most naturally occurring prenylated purine alkaloids are N6-prenylated adenine derivatives (e.g., plant cytokinins). Previously reported 3-prenylated purine alkaloids include triacanthine, saikachinoside A, and dioicine.