In neuroscience research, cell culture systems are essential experimental platforms. It is of great interest to explore in vivo-like culture substrates. We explored how basic properties of neural cells, nuclei polarization, phenotypic differentiation and distribution/migration, were affected by the culture at poly-L-lactic acid (PLLA) fibrous scaffolds, using a multipotent mitogen-expanded human neural progenitor cell (HNPC) line. HNPCs were seeded, at four different surfaces: two different electrospun PLLA (d = 1.2 - 1.3 μm) substrates (parallel or random aligned fibers), and planar PLL- and PLLA surfaces. Nuclei analysis demonstrated a non-directed cellular migration at planar surfaces and random fibers, different from cultures at aligned fibers where HNPCs were oriented parallel with the fibers. At aligned fibers, HNPCs displayed the same capacity for phenotypic differentiation as after culture on the planar surfaces. However, at random fibers, HNPCs showed a significant lower level of phenotypic differentiation compared with cultures at the planar surfaces. A clear trend towards greater neuronal formation at aligned fibers, compared to cultures at random fibers, was noted. We demonstrated that the topography of in vivo-resembling PLLA scaffolds significantly influences HNPC behavior, proven by different migration behavior, phenotypic differentiation potential and nuclei polarization. This knowledge is useful in future exploration of in vivo-resembling neural cell system using electrospun scaffolds.