Reports on metabolism of vitamin B6 in higher plants are scarce except for the partial purification of pyridoxine (pyridoxamine) 5'-phosphate oxidase from barley seedlings. Previously, we demonstrated that the main metabolite of pyridoxine (1) in etiolated plant seedlings is 5'-O-(β-D-glucopyranosyl)pyridoxine (2). In this study, we surveyed metabolites of 1 in seedlings of podded pea (Pisum sativum L. cv. Kinusaya) and found a small amount of a derivative (3) in addition to 2. The present paper describes the isolation and structure of 3. Seedlings of podded pea grown on a solution of 1 were extracted with hot 75% EtOH. Metabolite 3 was separated from 1 and 2 by adsorption on Dowex 1×4 (AcO⁻) and further purified by serial column chromatography on cellulose powder, Dowex 1×4 (AcO⁻), and P-cellulose (H⁺), yielding a colorless powder. The structure of 3 was elucidated using UV, IR, PMR, and ¹³C NMR spectroscopy, alkaline hydrolysis, and the Gibbs color reaction. Compound 3 showed the same UV absorption spectrum as 1 and 2 (λmax 292 nm in 0.1 N HCl) and IR bands for ester carbonyl and carboxyl groups. PMR and ¹³C NMR data indicated the presence of the 2 structure plus additional moieties. Alkaline hydrolysis of 3 produced 2 and 3-hydroxy-3-methylglutaric acid (4). The Gibbs reaction revealed unsubstituted hydroxyl groups at C-3 and C-4' of the pyridoxine moiety. ¹³C NMR chemical shift differences between 3 and 2 indicated the ester bond was at the C-12 hydroxyl of the glucose moiety of 2. Thus, the structure of 3 was determined to be 5'-O-[6-O-(3-hydroxy-3-methyl-4-carboxybutanoyl)-β-D-glucopyranosyl]pyridoxine. Compound 3 is formed from 1 in various Leguminosae seedlings and likely occurs naturally in plant tissues.