Innate immune pathways are early defense responses important for the immediate control and eventual clearance of many pathogens, where signaling is initiated via pattern recognition receptor (PRR)-mediated events that occur in a ligand- and cell-type specific manner. Within CNS neurons, innate immune pathways are likely crucial to control pathogens that target these essential yet virtually irreplaceable cells. However, relatively little is known about the induction and regulation of neuronal PRR signaling. In this report, we used human neuronal cell lines and primary rat neuronal cultures to examine PRR expression and function. We found that several innate immune receptor ligands, including Sendai virus, the dsRNA mimetic polyinosinic-polycytidylic acid, and LPS all activated differentiation-dependent neuronal innate immune pathways. Functional genetic analyses revealed that IFN regulatory factor 3-mediated pathways that resulted in IFN-beta transcriptional upregulation were activated in cultured human neuronal cells by the PRRs TLR3, MDA5, or RIG-I in a ligand-specific manner. Furthermore, genome-wide transcriptional array and targeted genetic and pharmacologic analyses identified PI3K signaling as crucial for the induction of innate immune pathways in neurons. These results indicate that human neuronal cells possess specific and functional PRR pathways essential for the effective induction of innate immune responses, and suggest that neurons can play an active role in defense against neurotropic pathogens.