Dietary polyphenols present in fruits and vegetables have been reported to manifest beneficial health effects on humans. Polyphenol metabolites including their sulfated derivatives have been shown to be biologically active. Primarily due to the difficulty in preparing regiospecific sulfated polyphenols for detailed investigations, the exact functions of sulfated polyphenols, however, remain unclear. The current study aimed to develop a procedure for the regioselective production of sulfated polyphenols using Escherichia coil cells expressing human cytosolic sulfotransferases (SULTs). Two regioisomers of sulfated genistein were produced by E. coli cells expressing human SULT1A3, SULT1C4, or SULT1E1, and purified using Diaion HP20 resin, followed by high pressure liquid chromatography (HPLC). Structural analysis using mass spectrometry (MS) and nuclear magnetic resonance (NMR) revealed that E. coli cells expressing SULT1A3 preferentially produced genistein 4'-sulfate, whereas E. coil cells expressing SULT1C4 preferentially produced genistein 7-sulfate. To improve the bioproductivity, the effects of several factors including the concentrations of glucose and SC42-, and growth temperature were investigated. The bioproduction procedure established in this study will be valuable for the production of regioselective sulfated polyphenols for use in future studies on their biological functions. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.