Understanding the extracellular matrix (ECM) effect on pancreatic β cells is critical to optimise the derivation of functional β cells for transplantation and understand mechanisms that control islet neogenesis and glucose homeostasis. We assessed the effect of natural ECMs [collagen I, collagen IV, laminin and fibronectin (FN)] on rat islets of Langerhans' morphology, adhesion, viability, functionality and islet specific genes expression after 7 days in vitro culture. However, we could not detect a significant difference on the other parameters in these ECMs and islets interaction. To examine islets interactions, we used a synthetic three-dimensional surface composed of electrospun polyamide nanofibres. Laminin-coated nanofibrillar surfaces, but not laminin or nanosurface alone, induced comparable expression of the Ins1 and Ins2 genes in adult β cells. Using a glucose challenge test, a marked response of insulin secretion by islets occurred that were cultured on laminin-coated nanofibrillar surfaces. We contend that the reestablishment of cellular interactions by the combination of nanomaterials and natural ECMs can be useful in maintaining in vitro islet functions.