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Biologically functional beta-cells proliferate at an extremely low rate with limited turnover capacity. This cellular property hinders cell-based therapy for clinical applications. Many attempts have been made to develop techniques that allow large quantities of production of clinically relevant islet β-cells in vitro. A line of studies demonstrates that functional beta-cells can proliferate under certain circumstances, providing hope for generating and expanding these cells in vitro and transplanting them into the recipient. In this study, we showed that a membrane substrate offers a better niche for beta cell proliferation and insulin secretion. Mouse beta cells were grown on a tissue culture plate (TCP) as a control as well as on polyethylene terephthalate (PET) membrane, and cell numbers were counted four times at 48 hours intervals. The cell doubling time was shortened from 64.7±0.4 h for beta cells grown on TCP, to 38.6±0.5 h (p-value 0.03) for those grown on PET membrane substrate with a pore size of 1µm. In addition, there was an increase of approximately ten to thirteen fold in insulin gene expression in cells cultured on PET compared to that on TCP (p-value 0.02). Furthermore, to investigate the mechanism of the enhanced proliferation and insulin production using membrane substrate, the expression profile of eighty-four genes that are involved in the apoptotic pathway were measured by quantitative real time polymerase chain reaction (qRT-PCR). Enhancements in Akt and Bcl2 gene expression were detected. These findings demonstrate that a membrane substrate can offer better physicochemical cues for enhancing β-cells proliferation and function in vitro.