Selective separation of specific microbial types from a heterogeneous microbial population, such as an environmental microbial community, is an important process for microbial research and biotechnological industries. In the present study, pH-gradient ion-exchange microbial cell chromatography (PIE-MCC) was developed as a new method for microbial separation. The proposed method enables target microorganisms to be separated from a microbial community based on differences in microbial surface characteristics, because these characteristics, such as the zeta (zeta)-potential, vary among microbial cells. PIE-MCC was conducted by controlling the adhesion and detachment of microbial cells to and from the carrier surface by manipulating the pH of the running buffer. As a proof of concept, microbial cell separation via PIE-MCC was demonstrated using pure-cultured strains, model mixtures of two different pure-cultured strains, and an environmental sample targeting uncultivated microorganisms; i.e., each pure-cultured strain showed unique chromatograms; specific single species were separated from the model mixture; and a specific, uncultivated target was separated from the environmental sample. The zeta-potential of several tested strains suggested that not only electrostatic interactions, but also other factors affected microbial adhesion to the carrier surface. The newly developed method has several potential advantages compared with other techniques, not only in terms of its microbial separation capability, but also in terms of its simplicity and ability to be scaled up. Thus, the method has the potential to be widely used for a variety of purposes in the microbiology and biotechnology fields. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.