In vitro models of human small intestine play a critical role in predicting oral drug absorption efficiency as well as understanding intestinal epithelia functionality. Conventional 2D in vitro models fail to represent small intestinal villi which are important 3D features for the small intestinal microenvironment. Without these features, in vitro models of the small intestine lack physiological relevance. By using microfabrication techniques, we created a plastic template with accurate dimensions and densities of human small intestinal villi. Based on this template, we fabricated collagen villous scaffolds utilizing a sacrificial hydrogel technique. By integrating collagen villous scaffolds into a custom designed insert kit, we developed the first 3D in vitro model of the human small intestine. Epithelial cells cultured on this 3D model presented unique cell differentiation morphology similar to human small intestine epithelia. Drug permeability tests conducted on this 3D model provided more accurate results for drugs absorbed through the paracellular pathway when compared to conventional 2D models. In addition, alternative biomaterials were tested for their potential in making 3D villous scaffolds. This work suggests that the 3D in vitro model of the human small intestine is an effective tool for predicting drug permeability as well as investigating the small intestinal microenvironment.