An important challenge in neuronal tissue engineering is to create innovative tools capable of promoting cellular response in terms of neuronal differentiation and neurite orientation that may be used as investigational platforms for studying neurobiological events and neurodegenerative disorders. A novel membrane bioreactor was created to provide a 3D well-controlled microenvironment for neuronal outgrowth. The bioreactor consisted of poly-L-lactic acid highly aligned microtube array (PLLA-MTA) membranes assembled in parallel within a chamber that establish an intraluminal and an extraluminal compartment whose communication occurs through the pores of the MTA membrane walls. The bioreactor configuration provided a wide surface area for cell adhesion in a small volume, and offered a peculiar arrangement that directed neuronal orientation. The combination of an appropriate membrane porosity, pore interconnectivity and very thin walls ensured optimal indirect perfusion to cell compartment, and enhanced the mass transfer of metabolites and catabolites protecting neurons from shear stress. The PLLA-MTA membrane bioreactor promoted the growth and differentiation of SH-SY5Y cells toward a neuronal phenotype, and guided neurite alignment giving rise to a 3D neuronal tissue-like construct. It provides an innovative platform to study neurobiological phenomena in vitro and by guiding neuronal orientation for repair and/or regeneration.