This study describes a technique for micropatterning rat hepatocytes using a micro stencil. A poly-dimethylsiloxane (PDMS) microstencil, in which a thin membrane comprising 724 through-microholes (500 μm in diameter) in a triangle arrangement of 800 μm pitch was sealed onto the surface of glass plate (24 × 24 mm), was used as a microstencil chip. The surface of the microstencil chip was coated with type IV collagen in order to encourage cell adhesion. Rat hepatocytes micropatterned on the chip by the stencil were peeled off from the chip. The micropatterned conformation of rat hepatocytes on the chip was maintained for at least 5 days of culture. Furthermore, the albumin section and ammonia removal activities of hepatocytes in the micropatterned culture were higher than those in randomly distributed culture. This simple technique for a micropatterned culture may be applicable as a cellular platform for cell-based assay systems.