In our previous study, we reported a miniaturized electrical field flow fractionation device (í-EFFF) that used a pulsed voltage (PV) to increase the effective electric field and, hence, improved the separation performance. In this work, we developed two í-EFFFs with planar or segmented electrode design and investigated the particle movement in the flow channels under a PV. Numerical simulation was used to understand the electric field distribution in the í-EFFFs. When the calculations for the í-EFFF with a segmented electrode (segmented í-EFFF) and the í-EFFF with planar electrodes (planar í-EFFF) are compared, a stronger electric field at the top electrode segments is found in the segmented í-EFFF, with the strongest field at the edges of the electrode segments. Nanoparticle motion in both devices was in situ visualized by using a fluorescence microscope equipped with a CCDcamera. Results reveal that electrophoresis governs the nanoparticle movement in the planar í-EFFF and dielectrophoresis dominates the movement in the segmented í-EFFF. Two models are postulated to explain the experimental observations of the nanoparticle movement. The mechanistic understanding of controlling nanoparticle motion in a miniaturized environment will help the design and application of í-EFFF for the separation of charged biomolecules (proteins and DNAs).