A bio-inspired in vitro disease model was developed to investigate the basic biophysics of atherosclerotic diseases. In vivo study was conducted in advance using zebrafish fed with a normal diet and a cholesterol-enriched diet. The endothelial cells (ECs) of the zebrafishes fed with a normal diet are tightly attached and aligned. Their collagen has a fiber-like structure. By contrast, the endothelial cells of the zebrafish on high cholesterol diet are disorganized and their collagen has broken structures. In vitro models of human umbilical vein endothelial cells (HUVECs) were established on collagen films to mimic such in vivo experimental results. The normal collagen film simulates the extracellular matrix (ECM) in the blood vessels of a normal zebrafish, and the collagenase-treated collagen film mimics the ECM in blood vessels of an abnormal zebrafish. The HUVECs cultured on the normal collagen film are tightly attached, similar to those of a normal zebrafish. However, the cells cultured on the collagenase-treated collagen film are aggregated and biomarkers of endothelial dysfunction are expressed on the surface of the abnormal endothelial cells established on the denatured collagen film. The present in vitro model using a bio-inspired collagen film has a great potential for the design of novel therapies for clinical treatments of atherosclerosis through better understanding on the outbreak mechanism of atherosclerosis.