This paper presents a microfluidic system for manipulation and separation of micron-sized particles based on the combined use of negative dielectrophoresis (DEP) and hydrodynamic forces. A 3-D microelectrode structure (so called paired electrode array) are constructed face to face on the top and bottom sides of the microchannel and driven with highfrequency AC voltage to generate dielectrophoretic gates. Depending on the relative strengths of the two forces, particles such as polystyrene beads or cells carrying by a laminar flow can either penetrate the gate or settle there. This gives rise to certain applications including selectively concentrating particles from the flow, separating particles depending on their sizes or dielectric properties, and automatically positioning particles to selective locations. For this purpose, a microfluidic device consisting of the paired electrode array sitting on the channel has been fabricated using microfabrication techniques. Polystyrene beads were used to study the performance of the device. Experimental results including the concentration and separation of particles are presented.