The present work reports the constants of pyridoxine (vitamin B-6) protolytic and tautomeric equilibria determined by means of potentiometry and spectrophotometry respectively in wide range of binary solvent (aqueous ethanol) compositions. The Gibbs energies of pyridoxine molecular and zwitterionic forms transfer from water to aqueous ethanol were obtained using method of partition between immiscible phases. The dependences of equilibrium processes constants on mixed solvent composition were analyzed taking into account the reagents solvation data. The transmembrane transport capability of B-6 vitamin was discussed. The effect of binary solvent composition on pyridoxine reactivity towards nucleophilic substitution reactions including phosphorylation reaction was estimated by quantum chemical calculations. (C) 2016 Elsevier Ltd. All rights reserved.