The electrokinetic focusing and the resultant accelerated electrophoretic motion of polystyrene particles and red blood cells were visualized in microfluidic cross-channels. The experimentally measured width of the focused stream and the measured velocity increase of particles and cells at different voltage ratios follow the proposed analytical formula within the experimental error. The attained velocity increase is insensitive to the particle size, particle property (i.e., particle or cell), and particle trajectory. By solving the electrical potential field in the cross-channel at the experimental conditions, we demonstrate that the squeezed electrical field lines in the channel intersection determine the shape of the focused stream, and the nonuniform distribution of axial electrical field strength underlies the variation of particle/cell electrophoretic velocity through the focusing region. However, the dielectrophoretic force resulting from the nonuniform electrical field in the intersection seems to push the acceleration region of particles and cells slightly in the downstream direction. We have also achieved the single particle/cell dispensing by instantly triggering an electrical pulse perpendicular to the focused particulate flow in a double-cross microchannel. The electrokinetic manipulation of particle/cell in microchannels demonstrated in this work can be used for developing integrated lab-on-a-chip devices for studies of cells.