MYB transcription factors are a large family of proteins involved in the regulation of secondary metabolism and cell shape, the enhancement of disease resistance and the response to different stresses. In this study, the role of TaMYB4 in wheat against biotic and abiotic stresses was investigated. TaMYB4 was cloned from wheat cv. Suwan11 [the leaves were infected with Puccinia striiformis f.sp. tritici (Pst)]; the TaMYB4 protein is 243 amino acids in length. In addition, TaMYB4 exhibited high similarity with BdMYB4 from Brachypodium distachyon, which was also identified as a member of the R2R3-MYB family of genes. Furthermore, transient expression analysis showed that the deduced TaMYB4 protein was localised in the nucleus of onion epidermal cells. Additionally, a yeast one-hybrid assay revealed that TaMYB4 exhibits transcriptional activity and the C-terminus is necessary for the activation of transcription. The transcript levels of TaMYB4 were observed directly and were found to be significantly upregulated in the early stage and 48 h after inoculation with the incompatible Pst. The transcripts of TaMYB4 were detected in the wheat roots, culms and leaves. Moreover, the transcription of TaMYB4 was induced by salicylic acid, ethylene, abscisic acid and methyl jasmonate hormones. The same results were obtained with cold and wound treatments. Furthermore, the knockdown of TaMYB4 expression using virus-induced gene silencing enhanced the susceptibility of wheat cultivar Suwon11 to the incompatible race of Pst. These results demonstrate that TaMYB4 plays a role in the wheat response to biotic stress.