Thispaper reports a novel method to concentrate bacteria in three-dimension by negative dielectrophoretic (n-DEP) force in a microchannel. This was achieved by placing a thin dielectric layer on one of a pair of parallel plate electrodes. The dielectric layer having a home-plate like pentagonal shape, forms a gradient of electric field causing n-DEP. A three-dimensional numerical simulation of bacteria trajectory predicts that bacteria flowing a microchannel were three-dimensionally concentrated beneath the tip of the pentagonal dielectric thin layer. The trajectory and concentration of bacteria under n-DEP force were also experimentally confirmed using Escherichia coli cells. Bacteria moved along edges of the dielectric layer and were pushed to the opposite electrode, resulting in their concentration in three-dimension. The proposed device might be applicable to selective concentration of bacteria depending on their dielectric properties.