This study identifies a novel class of small RNAs, tRNA-derived RNA fragments (tRFs), which are distinct from microRNAs (miRNAs) and siRNAs. tRFs are generated from tRNAs through precise processing at the 5' or 3' ends, forming three series: tRF-5, tRF-3, and tRF-1. These tRFs are the second most abundant class of small RNAs after miRNAs and exhibit specific expression patterns and biological roles. The authors demonstrate that tRF-1001, a member of the tRF-1 series, is highly expressed in cancer cell lines and negatively regulates cell proliferation. knockdown of tRF-1001 impairs cell proliferation, and this effect is rescued by introducing a synthetic 2'-O-methyl tRF-1001 oligoribonucleotide. tRF-1001 is generated in the cytoplasm by the tRNA 3'-end nuclease ELAC2, a prostate cancer susceptibility gene. These findings suggest that tRFs are not random by-products of tRNA degradation but have specific functions in cellular processes.This study identifies a novel class of small RNAs, tRNA-derived RNA fragments (tRFs), which are distinct from microRNAs (miRNAs) and siRNAs. tRFs are generated from tRNAs through precise processing at the 5' or 3' ends, forming three series: tRF-5, tRF-3, and tRF-1. These tRFs are the second most abundant class of small RNAs after miRNAs and exhibit specific expression patterns and biological roles. The authors demonstrate that tRF-1001, a member of the tRF-1 series, is highly expressed in cancer cell lines and negatively regulates cell proliferation. knockdown of tRF-1001 impairs cell proliferation, and this effect is rescued by introducing a synthetic 2'-O-methyl tRF-1001 oligoribonucleotide. tRF-1001 is generated in the cytoplasm by the tRNA 3'-end nuclease ELAC2, a prostate cancer susceptibility gene. These findings suggest that tRFs are not random by-products of tRNA degradation but have specific functions in cellular processes.