Until recently, transfer RNAs (tRNAs) were thought to function in protein translation only. However, recent findings demonstrate that both pre‐ and mature tRNAs can undergo endonucleolytic cleavage by different ribonucleases originating different types of small non‐coding RNAs, known as tRNA‐derived fragments (tRFs). tRFs are classified according to their origin and are implicated in various cellular processes, namely apoptosis, protein synthesis control, and RNA interference. Although their functions are still poorly understood, their mechanisms of action vary according to the tRF sub‐type. Several tRFs have been associated with cancer, neurodegenerative disorders, and viral infections and growing evidence shows that they may constitute novel molecular targets for modulating pathological processes. Here, we recapitulate the current knowledge of tRF biology, highlight the known functions and mechanisms of action of the different sub‐classes of tRFs and discuss their implications in human disease. WIREs RNA 2017, 8:e1423. doi: 10.1002/wrna.1423This article is categorized under: RNA Processing > tRNA Processing Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease