Virus infection rapidly induces type-I interferon (IFN-α/β) and proinflammatory cytokines, which further activates a subset of genes that confer an antiviral state on the host cell, thereby constituting the first line of defense against invading viruses. Despite major advances over the last decade in our understanding of the innate immune signaling pathways, the human gene components involved in the pathways have not yet been fully elucidated. Here, we have begun to systematically identify additional pathway components by high-throughput cell-based screening of human cDNA libraries that represent about 50% of the human coding transcriptome. Using luciferase-based transcriptional reporter assays that measure the activation of IFN-β and/or NF-κB promoters, combined with data mining, we have been able to identify a number of novel positive and negative regulators in the pathways. Hit validities were confirmed by various biochemical, genetic and biophysical methods, including shRNA knockdown and/or knockout mouse-based methods, RT-PCR, immunoblotting and co-immunoprecipitation. We expect our findings will offer novel targets for therapeutic manipulation of the innate immune signaling pathways, leading to the development of more effective agents to treat virus- and inflammation-related diseases.