We present an engineered three-dimensional (3D) in vitro brain microvasculature system embedded within the bulk of a collagen matrix. To create a hydrogel template for the functional brain microvascular structure, we fabricated an array of microchannels made of collagen I using microneedles and a 3D printed frame. By culturing mouse brain endothelial cells (bEnd.3) on the luminal surface of cylindrical collagen microchannels, we reconstructed an array of brain microvasculature in vitro with circular cross-sections. We characterized the barrier function of our brain microvasculature by measuring transendothelial permeability of 40 kDa fluorescein isothiocyanate-dextran (Stoke's radius of ∼4.5 nm), based on an analytical model. The transendothelial permeability decreased significantly over 3 weeks of culture. We also present the disruption of the barrier function with a hyperosmotic mannitol as well as a subsequent recovery over 4 days. Our brain microvasculature model in vitro, consisting of system-in-hydrogel combined with the widely emerging 3D printing technique, can serve as a useful tool not only for fundamental studies associated with blood-brain barrier in physiological and pathological settings but also for pharmaceutical applications.