RIG-I-like receptors (RLRs) are key sensors of viral infection, mediating the transcriptional induction of type I interferons and other genes that establish an antiviral host response. Recent studies show that both viral and host-derived RNAs can trigger RLR activation, leading to effective antiviral responses or immunopathology if not controlled. This review discusses recent advances in understanding RLRs' roles in viral infection, malignancies, and autoimmune diseases, as well as how RLR activity is regulated by host mechanisms, including RLR-interacting proteins, post-translational modifications, and non-coding RNAs. Key questions in the RLR field include translating RLR biology into new therapeutics. RLRs are RNA sensors localized in the cytosol and include RIG-I, MDA5, and LGP2. These proteins detect immunostimulatory RNAs, such as those from viral genomes, and activate signaling pathways that lead to type I interferon production. RIG-I and MDA5 have CARD domains that mediate downstream signal transduction, while LGP2 regulates RIG-I and MDA5. RLRs recognize specific RNA features, such as 5'-PPP groups and duplex structures, which are characteristic of viral RNAs but absent in most host RNAs. Viral RNAs, particularly those from negative-sense RNA viruses, activate RIG-I and MDA5. Host RNAs can also activate RLRs, as seen in herpesvirus infections, where cellular RNAs like 5S rRNA pseudogenes are recognized by RIG-I. RLR activation can occur in sterile conditions, such as in autoinflammatory and autoimmune diseases, due to mutations or RNA metabolism defects. RLRs are regulated by post-translational modifications, including ubiquitylation, phosphorylation, acetylation, and SUMOylation, which control their activity and signaling. Synthetic RLR agonists, such as poly(I:C) and 5'-PPP duplex RNAs, are being explored for therapeutic applications in cancer and vaccination. RLR regulation is also influenced by interacting proteins, such as TRIM25, which mediates K63-linked ubiquitylation of RIG-I. Deubiquitylating enzymes and other regulatory mechanisms help maintain immune homeostasis by controlling RLR activity. Overall, understanding RLR biology is crucial for developing new therapies targeting viral and autoimmune diseases.RIG-I-like receptors (RLRs) are key sensors of viral infection, mediating the transcriptional induction of type I interferons and other genes that establish an antiviral host response. Recent studies show that both viral and host-derived RNAs can trigger RLR activation, leading to effective antiviral responses or immunopathology if not controlled. This review discusses recent advances in understanding RLRs' roles in viral infection, malignancies, and autoimmune diseases, as well as how RLR activity is regulated by host mechanisms, including RLR-interacting proteins, post-translational modifications, and non-coding RNAs. Key questions in the RLR field include translating RLR biology into new therapeutics. RLRs are RNA sensors localized in the cytosol and include RIG-I, MDA5, and LGP2. These proteins detect immunostimulatory RNAs, such as those from viral genomes, and activate signaling pathways that lead to type I interferon production. RIG-I and MDA5 have CARD domains that mediate downstream signal transduction, while LGP2 regulates RIG-I and MDA5. RLRs recognize specific RNA features, such as 5'-PPP groups and duplex structures, which are characteristic of viral RNAs but absent in most host RNAs. Viral RNAs, particularly those from negative-sense RNA viruses, activate RIG-I and MDA5. Host RNAs can also activate RLRs, as seen in herpesvirus infections, where cellular RNAs like 5S rRNA pseudogenes are recognized by RIG-I. RLR activation can occur in sterile conditions, such as in autoinflammatory and autoimmune diseases, due to mutations or RNA metabolism defects. RLRs are regulated by post-translational modifications, including ubiquitylation, phosphorylation, acetylation, and SUMOylation, which control their activity and signaling. Synthetic RLR agonists, such as poly(I:C) and 5'-PPP duplex RNAs, are being explored for therapeutic applications in cancer and vaccination. RLR regulation is also influenced by interacting proteins, such as TRIM25, which mediates K63-linked ubiquitylation of RIG-I. Deubiquitylating enzymes and other regulatory mechanisms help maintain immune homeostasis by controlling RLR activity. Overall, understanding RLR biology is crucial for developing new therapies targeting viral and autoimmune diseases.