This paper considered electrophoretic motion of a sphere in an aqueous electrolyte solution in a microchannel under the gravitational field. In an externally applied electric field, the negatively charged sphere will move toward the anode. At the same time, the sphere will move toward the lower channel wall due to the density difference and the gravity. When the sphere moves very close to the lower wall, the buoyancy, the electric double layer interaction force, and the van der Waals force balance the gravity force, so the sphere moves parallel to the lower wall. A theoretical model for the electrophoretic motion of a sphere in a microchannel, with the consideration of the electrophoretic retardation effect, is presented in this paper. It was found that the sphere's motion in the microchannel is affected by its size, the density difference, the zeta potentials of the sphere and the channel wall, and the applied electric strength. The effects of these factors on the sphere's transport distance in the microchannel are discussed. It was found that the spheres with the same surface charge could be separated by their size within a certain range of ka in aqueous solutions in the microchannel.