In neuronal tissue engineering many efforts are focused on creating biomaterials with physical and chemical pathways for controlling cellular proliferation and orientation. Neurons have the ability to respond to topographical features in their microenvironment causing among others, axons to proliferate along surface features such as substrate grooves in micro-and nanoscales. As a consequence these neuronal elements are able to correctly adhere, migrate and orient within their new environment during growth. Here we explored the polarization and orientation of hippocampal neuronal cells on nonpatterned and micro-patterned biodegradable poly(l-lactic acid) (PLLA) membranes with highly selective permeable properties. Dense and porous nonpatterned and micro-patterned membranes were prepared from PLLA by Phase Separation Micromolding. The micro-patterned membranes have a three-dimensional structure consisting of channels and ridges and of bricks of different widths. Nonpatterned and patterned membranes were used for hippocampal neuronal cultures isolated from postnatal days 1–3 hamsters and the neurite length, orientation and specific functions of cells were investigated up to 12 days of culture. Neurite outgrowth, length plus orientation tightly overlapped the pattern of the membrane surface. Cell distribution occurred only in correspondence to membrane grooves characterized by continuous channels whereas on membranes with interconnected channels, cells not only adhered to and elongated their cellular processes in the grooves but also in the breaking points. High orientation degrees of cells were determined particularly on the patterned porous membranes with channel width of 20 μm and ridges of 17 μm whereas on dense nonpatterned membranes as well as on polystyrene culture dish (PSCD) controls, a larger number of primary developed neurites were distributed. Based on these results, PLLA patterned membranes may directly improve the guidance of neurite extension and thereby enhancing their orientation with a consequently highly ordered neuronal cell matrix, which may have strong bearings on the elucidation of regeneration mechanisms.