A cell separation device includes a channel or chamber in which a sample flows or moves, the sample including target cells marked with magnetic particles, and non-target cells, and a magnet which generates a magnetic first force in a first direction with respect to the sample within the channel or chamber. The channel or chamber of the cell separation device is applied with a second force in a second direction opposite to the first direction of the magnetic force. According to the cell separation device and a method of separating cells, the target cells move in the first direction by the magnetic force, and the non-target cells move in the second direction by the second force, by simultaneously applying the magnetic force and the second force in opposing directions, thereby separating the target cells from the non-target cells.