OBJECTIVE Production of genetically modified mice including Knock-out (KO) or Knock-in (KI) mice is necessary for organism-level phenotype analysis. Embryonic stem cell (ESC)-based technologies can produce many genetically modified mice with less time without crossing. However, a complicated manual operation is required to increase the number of ESC colonies. Here, the objective of this study was to design and demonstrate a new device to easily find colonies and carry them to microwells. RESULTS We developed a polydimethylsiloxane-based device for easy manipulation and isolation of ESC colonies. By introducing ESC colonies into the groove placed near culturing microwells, users can easily find, pick up and carry ESC colonies to microwells. By hydrophilic treatment using bovine serum albumin, 2-μL droplets including colonies reached the microwell bottom. Operation time using this device was shortened for both beginners (2.3-fold) and experts (1.5-fold) compared to the conventional colony picking operation. Isolated ESC colonies were confirmed to have maintained pluripotency. This device is expected to promote research by shortening the isolation procedure for ESC colonies or other large cells (e.g. eggs or embryos) and shortening training time for beginners as a simple sorter.