Extracellular matrix (ECM)-based hydrogels can serve as scaffolds in reconstruction of physiologically relevant threedimensional (3D) in vitro models. Biocompatible or integrated hydrogels can be used to modulate ECM properties such as stiffness and composition for studies of cell−ECM interactions and morphogenesis. To this end, we developed a new type of spatially modified collagen type 1 hydrogel by convective addition of collagen type 1 solution. The matrix displayed properties that were distinct from those of a collagen type 1 hydrogel and recapitulated the morphology and function of mammary epithelium in a 3D microfluidic platform. In this ECM, mammary epithelial cells secreted laminin and exhibited self-assembly into a basement membrane. Thus, this spatially modified ECM offers biophysical features that can facilitate the construction of mammary epithelium and, by extension, that of various other epithelial types. Additionally, our reconstructed mammary duct can be used as an in vitro model for the study of early-stage breast cancer.