Transcription factors (TFs) play crucial roles in the transcriptional regulation of plant development and defence responses. Increasing evidence has implicated ETHYLENE INSENSITIVE3 (EIN3) in the plant defence response to pathogen infection and environmental stimuli. However, the role of EIN3 in wheat resistance to Puccinia striiformis f. sp. tritici (Pst) is not clear. Here, TaEIL1 was isolated by rapid amplification of cDNA ends (RACE) based on a sequence fragment from a wheat-Pst interaction cDNA library. The TaEIL1 protein contains a typical EIN3-binding domain, and transient expression analyses indicated that TaEIL1 is localized in the nucleus. Yeast one-hybrid assay revealed that TaEIL1 exhibits transcriptional activity, and its C-terminus is necessary for the activation of transcription. TaEIL1 transcripts were regulated by environmental stress stimuli and were decreased under salicylic acid (SA) treatment. When wheat leaves were challenged with Pst, the transcript level of TaEIL1 in the compatible interaction was approximately three times higher than that in the incompatible interaction. Knocking down TaEIL1 through the Barley stripe mosaic virus (BSMV) virus-induced gene silencing (VIGS) system attenuated the growth of Pst, with shortened hyphae and reduced hyphal branches, haustorial mother cells and colony size. Moreover, enhanced necrosis was triggered by the Pst avirulent race CYR23, indicating that the hypersensitive response was strengthened in TaEIL1-silenced wheat plants. Thus, the up-regulation of defence-related genes and increased sucrose abundance might contribute to the enhanced disease resistance of wheat to the virulent race CYR31. Taken together, the results suggested that the suppression of TaEIL1 transcripts could enhance the resistance of wheat to stripe rust fungus.