The ability to control the behavior of living cells in vitro is critical in a wide range of research fields including fundamental cell biology, tissue engineering, and cell-based chip devices. Patterning cells on substrate surfaces is one of the most important approaches to modulate cellular behaviors and functions in culture. Here, we describe a cell patterning technique based on an electrochemical method, "electrochemical biolithography", which enables the localized immobilization of living cells under typical physiological conditions such as those found in culture media. The present technique is applicable even for a previously cell-patterned substrate and for a grooved substrate, opening new possibilities for the cell-based studies. Also, the integration of this electrochemical technique into a microfluidic device will provide a novel type of cell-chip which enables on-demand immobilization of cells just prior to the use of the device.