Transfer RNAs are the adaptors in protein synthesis that provide the key link between the nucleic acid-based genetic blueprint and proteins. While the central role of tRNAs in protein synthesis has been known for over 60 years, recent discoveries of their many non-canonical functions and therapeutic potential have heightened interest in tRNAs. In this thematic series, we highlight some of the developments presented at the recent biennial “International tRNA Workshop”. The topics chosen reflect advances that were enabled by the latest technological breakthroughs in structure determination and small RNA sequencing and emphasize the prospects and challenges of tRNA-based medicines to treat human diseases.