Producing sheet-like tissues is a promising strategy for implantable engineered tissues, because in vitro pre-vascularization is dispensable in this configuration. We developed a simple methodology for the formation and non-destructive harvesting of a thick pancreatic β-cell sheet consisting of mouse insulinoma MIN6-m9 cells and mouse NIH3T3 fibroblasts using an O2-permeable polydimethylsiloxane plate modified with poly(N-isopropylacrylamide) (O2+/PNIPA-PDMS plate). Owing to the direct oxygenation of the cells through the PNIPA-modified PDMS plate, a viable, metabolically active sheet 5-6 cell layers thick (ca. 60 μm thick) was formed spontaneously; in the absence of direct oxygenation, only a thin cell sheet could be formed consisting of at most 2 layers (ca. 20 μm thick) with mainly anaerobic metabolism. Consequently, the net density of MIN6-m9 cells under direct oxygenation was about twice as high as in the absence of direct oxygenation. Accordingly, the insulin secretion for 10 to 60 min after glucose stimulation was also about 1.5 times higher with oxygenation. Furthermore, the thick cell sheet was successfully harvested from the O2+/PNIPA-PDMS plate surface in a non-destructive manner by inducing a phase transition of PNIPA by lowering the temperature below the lower critical solution temperature. Thus, the present report shows a promising and simple method to produce thick sheet-like engineered tissues for transplantation that could be used as a treatment for type 1 diabetes.