New types of small RNAs distinct from microRNAs (miRNAs) are progressively being discovered in various organisms. In order to discover such novel small RNAs, a library of 17- to 26-base-long RNAs was created from prostate cancer cell lines and sequenced by ultra-high-throughput sequencing. A significant number of the sequences are derived from precise processing at the 5' or 3' end of mature or precursor tRNAs to form three series of tRFs (tRNA-derived RNA fragments): the tRF-5, tRF-3, and tRF-1 series. These sequences constitute a class of short RNAs that are second most abundant to miRNAs. Northern hybridization, quantitative RT-PCR, and splinted ligation assays independently measured the levels of at least 17 tRFs. To demonstrate the biological importance of tRFs, we further investigated tRF-1001, derived from the 3' end of a Ser-TGA tRNA precursor transcript that is not retained in the mature tRNA. tRF-1001 is expressed highly in a wide range of cancer cell lines but much less in tissues, and its expression in cell lines was tightly correlated with cell proliferation. siRNA-mediated knockdown of tRF-1001 impaired cell proliferation with the specific accumulation of cells in G2, phenotypes that were reversed specifically by cointroducing a synthetic 2'-O-methyl tRF-1001 oligoribonucleotide resistant to the siRNA. tRF-1001 is generated in the cytoplasm by tRNA 3'-endonuclease ELAC2, a prostate cancer susceptibility gene. Our data suggest that tRFs are not random by-products of tRNA degradation or biogenesis, but an abundant and novel class of short RNAs with precise sequence structure that have specific expression patterns and specific biological roles.