The RIG-I-like receptor (RLR) pathway is essential for detecting cytosolic viral RNA to trigger the production of type I interferons (IFNxcexb1/xcexb2) that initiate an innate antiviral response. Through systematic assessment of a wide variety of genomics data, we discovered 10 molecular signatures of known RLR pathway components that collectively predict novel members. We demonstrate that RLR pathway genes, among others, tend to evolve rapidly, interact with viral proteins, contain a limited set of protein domains, are regulated by specific transcription factors, and form a tightly connected interaction network. Using a Bayesian approach to integrate these signatures, we propose likely novel RLR regulators. RNAi knockdown experiments revealed a high prediction accuracy, identifying 94 genes among 187 candidates tested (~50%) that affected viral RNA-induced production of IFNxcexb2. The discovered antiviral regulators may participate in a wide range of processes that highlight the complexity of antiviral defense (e.g. MAP3K11, CDK11B, PSMA3, TRIM14, HSPA9B, CDC37, NUP98, G3BP1), and include uncharacterized factors (DDX17, C6orf58, C16orf57, PKN2, SNW1). Our validated RLR pathway list (http://rlr.cmbi.umcn.nl/), obtained using a combination of integrative genomics and experiments, is a new resource for innate antiviral immunity research. 