We fabricated a nanocomposite substrate made of nanofibers on micropillar arrays by photolithography, soft lithography and electrospinning for cell culture studies. This nanocomposite substrate combines the advantage of the extra-cellular matrix (ECM)-like surface morphology and high porosity and low stiffness of underneath supporting material. For neuronal culture studies, we used nanofibers of poly (lactic-co-glycolic acid) (PLGA) on high aspect ratio micropillars of polydimethylsiloxane (PDMS). Our results showed that primary hippocampal neurons on such a nanocomposite substrate have different cell morphology than on flat surfaces and they showed more electric activities. Thus, the nanofiber-micropillar composite substrates were shown to be useful for neuron culture studies.