This paper presents a novel dielectrophoresis-based microfluidic device incorporating round hurdles within an S-shaped microchannel for continuous manipulation and separation of microparticles. Local nonuniform electric fields are generated due to the combined effects of obstacle and curvature, which in turn induce negative dielectrophoresis forces exerting on the particle that transport throughout the microchannel electrokinetically. Experiments were conducted to demonstrate the controlled trajectories of fix-sized (i.e. 10 or 15x μm) polystyrene particles, and size-dependent separation of 10 and 15 μm particles by adjusting the applied voltages at the inlet and outlets. Numerical simulations were also performed to predict the particle trajectories, which showed reasonable agreement with experimentally observed results. Compared to other microchannel designs that make use of either obstacle or curvature individually for inhomogeneous electric fields, the developed microchannel offers advantages such as improved controllability of particle motion, lower requirement of applied voltage, reduced fouling, and particle adhesion, etc. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.