Tumour-bearing mice were created by transplanting cancerous cell sheets onto the subcutaneous tissue of the dorsal region, using luciferase gene-transfected mammary gland adenocarcinoma cells, 4T1-luc2, to investigate the tumourigenicity of the cell sheet relative to a conventional injection of cell suspension. Contiguous breast cancerous cell sheets were harvested from temperature-responsive culture dishes by reducing the temperature from 37 °C to 20 °C; the sheets were then transplanted onto the dorsal side of the mouse subcutaneous tissue, using a chitin-based supporting membrane. Cell suspensions obtained by trypsin digestion were subcutaneously injected into the dorsal region of mice. The tumour growth of the transplanted cancer cells was evaluated by the tumour volume and by the bioluminescence from luciferase-gene transfected cancer cells, using an in vivo imaging system. The cell sheet method improved the 4 T1-luc2 engraftment efficiency in living mouse tissues at the initial stage by 13-fold compared with that from injecting cell suspensions. On day 14 after the transplantation, the tumour formation at the transplanted area of cell sheet-transplanted mice also accelerated, and the mean tumour volume became 1116 mm3, which was 10 times larger than that in cell suspension-transplanted mice. The cell sheets engrafted on the recipient tissues efficiently due to the preserved extracellular matrix on their basal sides, such that cancer cells were supplied with sufficient oxygen and nutrients from the host tissues to develop tumour tissues. Therefore, cancerous cell sheet-based transplantation is a promising method for efficiently creating cancer-bearing mice. Copyright © 2013 John Wiley & Sons, Ltd.