Background and Objective: The innate immune system deploys various pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), to detect the invasion of pathogens and initiate protective responses. TLR7 and TLR8 are located within the endosome of immune cells and are activated by singlestranded RNAs (ssRNAs). In addition to foreign ssRNAs from bacteria and viruses, endogenous self-ssRNAs, such as microRNAs (miRNAs), have been shown to activate TLR7 and TLR8, but such endogenous ssRNA ligands have not yet been fully elucidated. This scientific knowledge gap is partly derived from the technical limitations of standard RNA-seq, particularly its inability to capture non-miRNA-short non-coding RNAs (sncRNAs) lacking 5'-phosphate and 3'-hydoroxyl ends. However, recent advances in our understanding of "previously-hidden" sncRNAs, captured by RNA-seq using T4 polynucleotide kinase-treated RNA samples, have widened the pool of candidate ssRNA molecules that could act as endogenous ligands of ssRNA-sensing immune receptors. This has indeed been exemplified in the recent finding that, during immune response, transfer RNA (tRNA)-derived sncRNAs in macrophages are packaged into extracellular vesicles (EVs), and those tRNA-derived extracellular (ex-) sncRNAs can function as endogenous ligands of TLR7 when delivered to the endosome of recipient cells. In this review, we highlight advances in our understanding of the functional roles of tRNA-derived ex-sncRNAs as emerging activators of endosomal TLRs. Methods:We searched PubMed for articles relevant to our topic and published prior to September 2022.Key Content and Findings: tRNA-derived ex-sncRNAs could act as endogenous modulators of the immune system by activating endosomal TLRs in diverse pathological conditions, ranging from bacterial infection to cancers and neurological disorders.Conclusions: tRNA-derived ex-sncRNAs may constitute a significant class of bioactive circulating RNAs, and they are promising candidates as biomarkers in various diseases. Further research is required to realize their full implication in human health and disease.