ABSTRACT In vitro intestinal models can provide new insights into small intestinal function, including cellular growth and proliferation mechanisms, drug absorption capabilities, and host‐microbial interactions. These models are typically formed with cells cultured on 2D scaffolds or transwell inserts, but it is widely understood that epithelial cells cultured in 3D environments exhibit different phenotypes that are more reflective of native tissue. Our focus was to develop a porous, synthetic 3D tissue scaffold with villous features that could support the culture of epithelial cell types to mimic the natural microenvironment of the small intestine. We demonstrated that our scaffold could support the co‐culture of Caco‐2 cells with a mucus‐producing cell line, HT29‐MTX, as well as small intestinal crypts from mice for extended periods. By recreating the surface topography with accurately sized intestinal villi, we enable cellular differentiation along the villous axis in a similar manner to native intestines. In addition, we show that the biochemical microenvironments of the intestine can be further simulated via a combination of apical and basolateral feeding of intestinal cell types cultured on the 3D models. Biotechnol. Bioeng. 2014;111: 1222–1232. © 2014 Wiley Periodicals, Inc. Porous PLGA scaffolds with villus features allow directional growth of intestinal epithelial cells along the crypt‐villus axis (A), with full coverage of cells after 7 days culture (B‐C). The 3D topography of these scaffolds enable cellular polarization and differentiation in a manner similar to native intestinal tissue, as reflected by changes in brush‐border enzyme expression and TEER. The authors expect this technology to provide an exciting new platform for more realistic studies of intestinal function.