Automated continuous individual cell transfer is a critical step in single-cell applications using microfluidic devices. Cells must be aspirated gently from a buffer before transferring to operation zone so as not to artificially perturb their biostructures. Vision-based manipulation is a key technique for allowing nondestructive cell transportation. In this paper, we presented a design for an automated single-cell loading and supply system that can be integrated with complex microfluidic applications for examining or processing one cell at a time such as the current nuclear transplantation method. The aim of the system is to automatically transfer mammalian donor (similar to 15 mu m) or oocyte (similar to 100 mu m) cells one by one from a container to a polydimethylsiloxane (PDMS) microchannel and then transport them to other modules. The system consists of two main parts: a single-cell suction module, and a PDMS-based microfluidic chip controlled by an external pump. The desired number of vacuumed cells can be directed into themicrofluidic chip and stored in a docking area. From the batch, they can be moved to next module by activating pneumatic pressure valves located on two sides of the chip. The entire mechanism is combined with monitoring systems that perform detection/tracking and control.