The actin cytoskeleton is involved in plant defense responses. However, the role of the actin-depolymerizing factor (ADF) family, which regulates actin cytoskeletal dynamics, in plant disease resistance, is largely unknown. Here, we characterized a wheat (Triticum aestivum) ADF gene, TaADF7, with three copies located on chromosomes 1A, 1B, and 1D, respectively. All three copies encoded the same protein, although there were variations in 19 nucleotide positions in the open reading frame region. Transcriptional expression of the three TaADF7 copies were all sharply elevated in response to avirulent Puccinia striiformis f. sp. tritici (Pst) infection, with similar expression patterns. TaADF7 regulated the actin cytoskeletal dynamics by targeting the actin cytoskeleton to execute actin binding/severing activities. When the TaADF7 copies were all silenced by virus-induced gene silencing, the growth of Pst hypha increased and sporadic urediniospores were observed, as compared with control plants, when inoculated with avirulent Pst. In addition, reactive oxygen species (ROS) accumulation and hypersensitive response (HR) were greatly weakened, whereas cytochalasin B partially rescued the HR in TaADF7-knockdown plants. Together, these findings suggest that TaADF7 likely contributes to wheat resistance against Pst infection by modulating the actin cytoskeletal dynamics to influence ROS accumulation and the HR. This article is protected by copyright. All rights reserved.