We report a spheroid forming lab-on-a-chip that can be used to control the size of three-dimensional hepatocyte spheroids by using micro-rotational flow. This device can form hepatic spheroids with diameters in the range 130-430 μm by varying the chamber diameter and the cell density. The developed lab-on-a-chip consisted of a polydimethylsiloxane-based micro-chamber produced by soft lithography and a perfusion system consisting of a reservoir, a dampener, a shredder channel, and a peristaltic pump to constantly circulate cell culture medium containing human hepatocellular liver carcinoma cells. First, the media were introduced into the chamber at a volumetric flow rate of more than 1.1 ml/min to generate stable micro-rotational flow around the entire area of the chamber. Second, cells were attracted toward the center of the chamber, aggregated and formed a spheroid by reducing the flow rate to 0.4 ml/min over a period of 2 min. The spheroid stayed in the chamber and continued to rotate in the center. Micro particle image velocimetry (micro PIV) revealed that the region where the spheroid was formed was confined by micro-rotational flow, hence trapping the spheroid in the chamber. Spheroids with diameters between 150 and 200 μm were successfully created with a standard deviation of less than 13.2 %. In addition, we succeeded in creating 12 spheroids at one time with reproducibility of 18.2 % by arraying micro-chamber. The proposed device is the first lab-on-a-chip that not only controls the spheroid size but also can constantly supply nutrition and oxygen.