Islet transplantation has provided proof of concept that cell therapy can restore normoglycemia in patients with diabetes. However, limited availability of islet tissue severely restricts the clinical use of the treatment. Thus, there is an urgent need to develop new strategies to generate an abundant source of insulin-producing cells that could be used to treat diabetes. A potential approach is the in vitro expansion of pancreatic beta cells obtained from cadaveric organ donors. However, when human beta cells are expanded in vitro, they dedifferentiate and lose the expression of insulin, probably as a consequence of pancreatic islet dissociation into single cells. We have studied whether reestablishment of cell-cell and cell-matrix relationships with a biomimetic synthetic scaffold could induce redifferentiation of expanded dedifferentiated beta cells. Cells isolated from human islet preparations were expanded in monolayer cultures and allowed to reaggregate into islet-like cell clusters (ICCs). Afterward, ICCs were embedded between two thin layers of the noninstructive self-assembling peptide (SAP), RAD16-I or RAD16-I functionalized with the integrin-binding motif RGD (RAD16-I/RGD) (R: arginine, G: glycine, D: aspartic acid), which was expected to promote cell-extracellular matrix interactions. ICCs cultured with RAD16-I were viable, maintained their cluster conformation, and increased in size by aggregation of ICCs, suggesting a self-organizing process. ICCs cultured in RAD16-I/RGD showed enhanced cell adhesion to RAD16-I matrix and reexpression of the beta cell-specific genes, Ins, Pdx1, Nkx6.1, and MafA. Redifferentiation was caused solely by bioactive cues introduced to the RAD16-I peptide since no differentiation factors were added to the culture medium. The results indicate that RGD-functionalized SAP in sandwich conformation is a promising three-dimensional platform to induce redifferentiation toward a beta cell phenotype and to generate insulin-expressing cells that could be used in diabetes therapy.