Abstract Fragments of mature tRNAs have long been considered as mere degradation products without physiological function. However, recent reports show that tRNA fragments (tRFs) play prominent roles in diverse cellular processes across a wide spectrum of species. Contrasting the situation in other small RNA pathways the mechanisms behind these effects appear more diverse, more complex and are generally less well understood. In addition, surprisingly little is known about the expression profiles of tRFs across different tissues and species. Here, we provide an initial overview of tRF expression in different species and tissues, revealing very high tRF-levels particularly in the primate hippocampus. We further modulated the regulation capacity of selected tRFs in human cells by transfecting synthetic tRF mimics (“overexpression”) or antisense-RNAs (“inhibition”) and identified differentially expressed transcripts based on RNAseq. We then used a novel k-mer mapping approach to dissect the underlying targeting rules, demonstrating that 5’ tRNA halves (5’ tRHs) silence genes in a sequence-specific manner, while the most efficient target sites align to the mid-region of the 5’ tRH and are located within the CDS or 3’ UTR of the target. This amends previous observations that tRFs guide Argonaut proteins to silence their targets via a miRNA-like 5’ seed match and suggests a yet unknown mechanism of regulation. Finally, our data suggests that some 5’ tRHs are also able to sequence-specifically stabilize mRNAs as upregulated mRNAs are also significantly enriched for 5’ tRH target sites.