<p>Caco-2 cells are derived from human colon cancer and have the ability to differentiate into human intestinal epithelial-like cells. The 2D in vitro intestinal model of Caco-2 cells cultured on a semi-permeable membrane is widely used in drug development and the evaluation of absorption functions. However, these intestinal models lack the structural characteristics of the small intestine in vivo, and the cell behavior is not properly controlled. Previous studies have reported that the microstructure of the villi and crypts on a small intestine-mimicking scaffold promotes Caco-2 differentiation; however, the effect of the nanostructure of the small intestine-mimicking scaffold on Caco-2 differentiation remains unclear. This study aimed to elucidate the effects of nanostructures on the small intestine mimetic scaffold in Caco-2 differentiation. We fabricated a decellularized small intestine in which the basement membrane nanostructure was altered through a subtractive process. Caco-2 cells were cultured on decellularized small intestine for 21&#xa0;days, and the differentiation of Caco-2 cells was assessed. The microvillus density of Caco-2 cultured on decellularized small intestine that retained the unique nanostructure of small intestinal basement membrane was significantly higher than that of Caco-2 cultured on decellularized small intestine that did not retain the unique nanostructure of small intestinal basement membrane. This indicates that nanostructures specific to the basement membrane of the small intestine enhanced Caco-2 cell maturation.</p>