In the pancreatic islet transplantation for curing diabetes mellitus, reaggregation of pancreatic islet single cells for making ‘islet spheroid’ is considered as a useful strategy in terms of islet viability and gene delivery into islet. However, during aggregation of islet spheroid, their insulin secretion pattern was not recovered to that of intact islets. We have developed a new cell-culture system using poly(dimethylsiloxane) (PDMS)-based concave-patterned film for aggregating islet spheroid. Here we optimized the effective formation of islets spheroid by using the concave-patterned film with different microwell diameter (300, 500, and 700-μm). Aggregated islet spheroids in different concave microwells showed same morphology with a limited size range and diameter dependency of microwell, which was similar to that of intact islet. However, islet spheroid in flat-bottomed dish (control) showed random aggregation with broad size distribution. Interestingly, insulin secretion pattern of islet spheroids in 300 and 500-μm concave patterned-film was same to that of intact islets, which was caused by the improved cell-to-cell interaction that was confirmed by electron microscope and strong phalloidin immunostain. However, islet spheroids in concave patterned-film with 700-μm diameter and flat-dish did not show normal insulin secretion in accordance with different glucose concentration, which might be caused by weak cell-to-cell interaction. On the other hand, the viability of islet spheroids in concave patterned-film was not affected during cultivation due to biocompatibility of PDMS polymer. Collectively, we demonstrated that PDMS-based concave-patterned film could be used for designing islet spheroids with improved cellular functionality and size uniformity to cure diabetes mellitus.