It is often necessary to prefocus particles and cells into a tight stream for subsequent separation and/or analysis in microfluidic devices. A DC electric field has been widely used for particle and cell focusing in insulator-based dielectrophoretic (iDEP) microdevices, where a large field magnitude, a high constriction ratio, and/or a long microchannel are usually required to enhance the iDEP effect. We demonstrate, in this work, an AC iDEP focusing technique, which utilizes a low-frequency AC electric field to generate both an oscillatory electrokinetic flow of the particle/cell suspension and a field direction-independent dielectrophoretic force for particle/cell focusing in a virtually "infinite" microchannel. We also develop a theoretical analysis to evaluate this focusing in terms of the AC voltage frequency, amplitude, and particle size, which are each validated through both experimental demonstration and numerical simulation. The effectiveness of AC iDEP focusing increases with the second order of electric field magnitude, superior to DC iDEP focusing with only a first-order dependence. This feature and the "infinite" channel length together remove the necessity of large electric field and/or small constriction in DC iDEP focusing of small particles.