Versican is one of the major extracellular matrix (ECM) proteins in the brain. ECM molecules and their cleavage products critically regulate the growth and arborization of neurites, hence adjusting the formation of neural networks. Recent findings have revealed that peptide fragments containing versican C-terminus (G3 domain) are present in human brain astrocytoma. The present study demonstrated that a versican G3 domain enhanced cell attachment, neurite growth, as well as glutamate receptor-mediated currents in cultured embryonic hippocampal neurons. In addition, the G3 domain intensified dendritic spines, increased the clustering of both synaptophysin and the glutamate receptor subunit GluR2 and augmented excitatory synaptic activity. In contrast, a mutated G3 domain lacking the epidermal growth factor (EGF)-like repeats had little effect on neurite growth and glutamatergic function. Treating the neurons with the G3-conditioned medium rapidly increased the levels of phosphorylated EGF receptor (pEGFR) and phosphorylated extracellular signal regulated kinase (pERK), indicating an activation of EGFR-mediated signaling pathways. Blockade of EGFR prevented the G3-induced ERK activation and suppressed the G3-provoked enhancement of neurite growth and glutamatergic function, but failed to block the G3-mediated enhancement of cell attachment. These combined results indicate that the versican G3 domain regulates neuronal attachment, neurite outgrowth and synaptic function of hippocampal neurons via EGFR-dependent and independent signaling pathway(s). Our findings suggest a role for ECM proteolytic products in neural development and regeneration.