Dielectrophoresis (DEP) has been widely used to focus and separate cells and particles in microfluidic devices. This work first demonstrates negative and positive dielectrophoretic focusing of particles in a serpentine microchannel by changing only the electric conductivity of the suspending fluid. Due to the channel turn-induced dielectrophoretic force, particles are focused to either the centerline or the sidewalls of the channel when their electric conductivity is lower (i.e. negative DEP) or higher (i.e. positive DEP) than that of the fluid. These distinctive dielectrophoretic focusing phenomena in a serpentine microchannel are then combined to implement a continuous separation between particles of different sizes and electric conductivities. Such separation eliminates the fabrication of in-channel microelectrodes or micro-insulators that are typically required in DEP-based separation techniques. A numerical model is also developed to predict the particle motion, and the simulation results agree reasonably with the observed particle focusing and separation behaviors.