Low proliferation capacity of corneal endothelial cells (CECs) and worldwide limitations in transplantable donor tissues reveal the critical need of a robust approach for in vitro CEC growth. However, preservation of CEC-specific phenotype with increased proliferation has been a great challenge. Here we offer a biomimetic cell substrate design, by optimizing mechanical, topographical and biochemical characteristics of materials with CEC microenvironment. We showed the surprising similarity between topographical features of white rose petals and corneal endothelium due to hexagonal cell shapes and physiologically relevant cell density (≈ 2000 cells/mm2). Polydimethylsiloxane (PDMS) substrates with replica of white rose petal topography and cornea-friendly Young's modulus (211.85 ± 74.9 kPa) were functionalized with two of the important corneal extracellular matrix (ECM) components, collagen IV (COL 4) and hyaluronic acid (HA). White rose petal patterned and COL 4 modified PDMS with optimized stiffness provided enhanced bovine CEC response with higher density monolayers and increased phenotypic marker expression. This biomimetic approach demonstrates a successful platform to improve in vitro cell substrate properties of PDMS for corneal applications, suggesting an alternative environment for CEC-based therapies, drug toxicity investigations, microfluidics and organ-on-chip applications.