In this study, electrospinning was performed to fabricate silk fibroin (SF) nanofibrous mats, which were used as substrates for in vitro culture of rat hippocampal neurons. The light and electron micrographs demonstrated that the electrospun SF nanofibrous mat supported the survival and growth of the attached hippocampal neurons. MTT assay and immunocytochemistry in couple with Western blot analysis respectively indicated there was no significant difference in both the cell viability and expression levels of some proteins, including GAP-43, MAP-2, NF, and beta-tubulin, between hippocampal neurons cultured in the electrospun SF nanofibrous mat extract and in plain neuronal medium. Our results indicated that electrospun SF nanofibrous mats were biocompatible to primary culture of hippocampal neurons without cytotoxic effects on the cell phenotype and functions, raising a potential possibility of using these mats for CNS therapeutic applications.