The advent of deep sequencing technology has unearthed entirely novel classes of small RNAs that are distinct from micro RNAs (miRNAs) and small interfering RNAs (siRNAs). One such class of small RNAs are those derived from transfer RNAs (tRNAs), commonly referred to as transfer RNA-derived fragments (tRFs). tRFs are processed from both the 5´and 3´end of the mature or primary tRNAs and some of these have been shown to possess the ability to repress global translation or specifically silence mRNA targets that have a complementary sequence. Our working hypothesis is that in highly proliferative cells, tRFs are a product of the inadvertent processing of tRNAs. We predict that tRFs then act to repress translation, and therefore proliferation, in a negative feedback loop. We aim to unravel the mechanisms that tRFs use to mediate this translational control. To do this, we used deep sequencing, RNA immunoprecipitation, PCR and polysome profiling techniques to identify the binding partners of two different sub-classes of tRFs: (i) 5’tRFs and (ii) tRF-piRNAs in human cancer cell lines to infer how these may mediate interactions with the translational machinery. The first of these tRFs sub-classes included a 19-mer 5’tRF derived from the glutamate tRNA (tRF_Gln19), which is highly expressed in HeLa cells and appears to associate with the multi-synthetase complex (MSC); a collection of aminoacyl-tRNA synthetases responsible for channelling translation. A second sub-class of these tRFs exhibits an extremely high affinity for the Argonaute protein Hiwi2 in MDAMB231 cells, and are similar to piRNAs in length (27-32 nt); and so are named tRF-piRNAs. tRF-piRNAs exhibit miRNA-like abundances and appear to associate with chaperone proteins as well as actively translating ribosomes in the cytoplasm. Here we present evidence that tRFs directly target the translational pathway in order to perform their function.