— We propose a new framework and novel visual control system for motile cells in three-dimensional (3-D) space. Our goal is to utilize microorganisms as micro-robots in various applications by exploiting galvanotaxis (locomotor response to electrical stimulus) to actuate them. This requires automated motion control of swimming cells in 3-D space; in contrast, our previous work has been limited to 1-D or 2-D motion control on the focal plane. The system is capable of 3-D tracking and control of swimming cells by utilizing a high-speed vision system. A combination of “lock-on” tracking within the focal plane and automated focusing using a Depth-From-Diffraction method executed at 1-kHz frame rate ensures both detailed measurement and a large working space. Experimental results for closed-loop 3-D motion control of Paramecium cells trapped within a small 3-D region demonstrate the possibility of using microorganisms as micromachines.