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Small intestinal villi are projective microstructures from the mucosa that provide a large surface area for digestion and absorption. On the mucosa, intestinal epithelial cells undergo terminal differentiation in space-along the crypt-villus axis-until they slough off into the lumen. Despite this unique physiological feature, to date in vitro cultivation of the intestinal epithelial cells is routinely done at the planar tissue-culture surface. In this research, we fabricated a projective, three-dimensional (3-D) tissue-culture environment to provide a physiologically relevant condition for establishing the enterocyte cell culture in vitro. We used the mouse small intestinal epithelium as the model and applied a microfabrication process, UV-LIGA, to generate an array of microneedles with a similar projective structure and size (height: 400 μm, base: 135 μm in diameter) as those of the duodenal villi. In addition, we shaped the LIGA-derived poly (lactic acid) microneedles by acetone/ethanol erosion to create a smooth tip structure for the engraftment of human Caco-2 enterocytes. The engineered villus array had a total surface area of 4.81 cm2 per sq.cm. of planar surface, which led to a 2.48-fold increase in the cell number of enterocytes on the 3-D construct relative to that on the planar control surface. Staining tests of cellular components (nuclei and membranes), viability, and the ZO-1 tight-junction protein show that the projective PLA villus structure, similar to the two-dimensional surface, provided a suitable environment for the Caco-2 culture. This is the first time in UV-LIGA research field to use the 3-D lithography method to generate microstructures mimicking the intestinal structures. In addition, our work presents an initial step toward constituting a physiological gut in vitro by using an engineering approach for large-scale preparation of the biomimetic small intestine.