Ischemic injuries will lead to necrotic tissue damage and post-ischemia angiogenesis plays critical roles in blood flow restoration and tissue recovery. Recently, several types of small RNAs have been reported to be involved in this process. In this study, we first generated a rat brain ischemic model to investigate the involvement of new types of small RNAs in ischemia. We utilized deep sequencing and bioinformatics analyses to demonstrate that the level of small RNA fragments derived from tRNAs strikingly increased in the ischemic rat brain. Among these sequences, tRNA^Val- and tRNA^Gly-derived small RNAs account for the most abundant segments. The up-regulation of tRNA^Val- and tRNA^Gly-derived fragments was verified through northern blot and quantitative PCR analyses. The levels of these two fragments also increased in a mouse hindlimb ischemia model and cellular hypoxia model. Importantly, up-regulation of the tRNA^Val- and tRNA^Gly-derived fragments in endothelial cells inhibited cell proliferation, migration and tube formation. Furthermore, we showed that these small RNAs are generated by angiogenin cleavage. Our results indicate that tRNA-derived fragments are involved in tissue ischemia and we demonstrate for the first time that tRNA^Val- and tRNA^Gly-derived fragments inhibit angiogenesis by modulating the function of endothelial cells.