Abstract tRNA derived RNA fragments (tRFs) is an emerging group of small RNAs as abundant as miRNAs, and yet their roles are not well understood. Here, we focus on endogenous tRFs (18-22 bases) derived from 3’ end of human mature tRNAs (tRF-3) and their functions in gene repression. tRF-3 levels increase upon parental tRNA over-expression or tRNA induction by c-Myc oncogene activation. Elevated tRF-3 levels lead to repression of target genes with a sequence complementary to the tRF-3 in the 3’ UTR. The tRF-3-mediated repression is Dicer-independent, Argonaute-dependent and the targets are recognized by 5’ seed sequence rules similar to miRNAs. Furthermore, tRF-3s associate with GW proteins in P-bodies. RNA-seq identifies the endogenous target genes of tRF-3s that are specifically repressed upon tRF-3 induction. Overall, our analysis shows Dicer-independent tRF-3s, generated upon tRNA upregulation such as c-Myc overexpression, regulate gene expression globally through Argounate via seed sequence matches.