The structure of aloenin, a bitter glucoside from Aloe arborescens Mill. var. natalensis Berger which is used in domestic medicine, has been reinvestigated by a combination of the chemical and spectroscopic methods, and we now wish to revise the structure as 6-(2'-β-D-glucopyranosyloxy-4'-hydroxy-6'-methyl)phenyl-4-methoxy-2-pyrone (II). The biosynthetic pathway of this compound is of considerable interest because of its "masked polyketide structure." The ^<14>C-labeling pattern in aloenin (II) biosynthesized from [2-^<14>C]malonate, [1-^<14>C]acetate, [1-^<14>C]- and [3-^<14>C]phenylalanine, and [methyl-^<14>C]-methionine in Aloe plant demonstrated that the carbon skeleton of aloenin is generated by the acetate-malonate pathway and that the methyl of the methoxyl group is originated from methionine.