Fusarium head blight (FHB) caused by Fusarium graminearum is an important disease of wheat. FHB epidemics can cause tremendous yield losses, and also have negative impacts on human and animal health due to deoxynivalenol (DON) contamination. F. graminearum-wheat interaction is very complex and the mechanism remains largely unknown. tRNA-derived fragments (tRFs) are RNase-dependent small RNAs derived from tRNAs. Here we report tRFs are involved in wheat-F. graminearum interactions. Small RNAs from the spikelets inoculated with F. graminearum and mock from an FHB-susceptible variety Chinese Spring (CS) and an FHB-resistant variety Sumai3 (SM) were sequenced respectively. A total of 1,249 putative tRFs were identified. More tRFs were accumulated in FHB- susceptible variety CS than that in FHB-resistant variety SM, among which tRFGlu, tRFLys, tRFMet and tRFThr were dramatically induced by F. graminearum infection, with significantly higher fold changes in CS than those in SM. The expression patterns of the four highly induced tRFs were further validated by stem-loop qRT-PCR. The accumulation of tRFs was closely related to ribonucleases T2 family members, which were also induced by pathogen to degrade host tRNA to fragments of various sizes, and these tRFs could be assembled with AGO protein to form RNA-induced silencing complex (RISC) to inhibit the host gene expressions. In addition, during infection of the wheat cells, F. graminearum secreted DON to attack wheat ribosome and inhibit the formation of peptide chain, consequently leading to accumulation of tRNA. Therefore, tRFs might negatively regulate wheat resistance to FHB by interfering with the normal cell metabolism, cell cycle, and some of the disease resistance genes at post transcriptional level. The tRFs and their predicted targets identified here could be manipulated to enhance wheat resistance to FHB in breeding program.