We describe a method to create cell-adhesive regions and to position adherent cells on the newly created regions in sequence within a microfluidic channel. One of the microelectrodes fabricated at the channel wall was used for locally electrogenerating hypobromous acid that renders the opposite face of the channel protein-adsorptive. After the fibronectin was immobilized on the treated region, an ac voltage (1 MHz, 20 Vpp) was applied to the microelectrodes array in the presence of suspended HeLa cells. Since a repulsive force of negative dielectrophoresis (DEP) directs the cells toward the weakest region of the nonuniform electric field, the cells were positioned on the fibronectin-patterned region to allow the cell adhesion, even in the presence of fluid flow (<0.1 µL min⁻¹). By repeating the above process, two types of cells could be patterned in the microchannel.