Blufensins (Bln) have important functions in plant responses to biotic and abiotic stresses. Yet the precise functioning of Bln in the interaction between wheat (Triticum aestivum L.) and Puccinia striiformis f. sp. tritici (Pst) is still largely unknown. In this study, we isolated a stress-responsive Bln gene (TaBln4) from wheat. The expression of TaBln4 increased in host plants in the early stage after being infected with a virulent race (CYR31) of the Pst, but went unchanged in response to the avirulent Pst (CYR23). Furthermore, the transcriptional levels of TaBln4 were regulated by various hormone and abiotic stresses. Expression of TaBln4 in tobacco leaves blocked Bax-induced cell death, and knockdown of TaBln4 by virus-induced gene silencing (VIGS) inhibited host colonization by CYR31. Moreover, our detailed histological analyses showed that silencing of TaBln4 enhanced resistance to Pst by inducing a significant increase in the accumulation of reactive oxygen species (ROS) and the area of hypersensitive response (HR). Results from the BiFC and pull down assays demonstrate that TaBLN4 can interact with calmodulin. Thus, altogether, our results suggest that TaBln4 negatively regulates wheat resistance to Pst in an ROS- and HR-dependent manner.