In this study, the impact of randomly oriented electrospun polyamide nanofibrous architecture on neurogenic differentiation of human embryonic stem cells (hESCs) compared with the lack of nanofibrous features in vitro in a neural-inducing condition was examined. Flow cytometry analysis of hESC-derived neural ectoderm (NE) showed nanofibrous surfaces capable of supporting NE by expression of higher percentages of related markers NESTIN, SOX1, and PAX6 in addition to significantly greater total cell proliferation as shown by Ki67 in the neurogenic condition. After replating hESC-derived NE, the differentiated cells expressed higher neuronal markers (TUJ1 and MAP2) and motor neuron markers (HB9, ISL1, and ChAT) at both the protein and mRNA levels on nanofibers. The presence of developed spread neurites and plausible neurite connections were shown by scanning electron microscopy. Additionally, Na(+) and Ca(2+) currents in differentiated neurons on nanofibers were significantly greater than both control and generated action potentials. Moreover, less duration of inward currents, greater negative resting membrane potential, and enhanced expression and functionality of ionic channel genes were observed in neuronal cells on nanofibers. These results indicated that a nanofibrillar surface along with neurogenic growth factors provided a better environment for hESC neurogenic differentiation and function, which holds great promise in prospective tissue engineering applications.