Neurofascin is a member of the immunoglobulin superfamily involved in axon extension and fasciculation. Here we apply adenoviral short hairpin RNA (shRNA) expression in primary neurons, PC12–NIH/3T3 co-cultures in combination with Luminex® assays, to demonstrate homophilic interactions of neurofascin for neurite outgrowth. An adenoviral vector was constructed for the expression of shRNA in primary tectal cells that inhibits gene expression similar to short interfering RNA. We demonstrate that after shRNA-mediated knockdown neuronal neurofascin expression is important for neurite outgrowth on a neurofascin substrate. Neurite outgrowth assays reveal that neurite formation of PC12 cells is increased when neurofascin is overexpressed on both outgrowing PC12 cells and substrate NIH/3T3 cells, suggesting that neurofascin expression is also sufficient for neurite induction. Luminex technology for the analysis of protein–protein interactions showed homophilic binding of neurofascin to itself.