Fusarium head blight (FHB) is a devastating disease of wheat worldwide. Fhb1 is the most consistently reported quantitative trait locus (QTL) for FHB resistance breeding. A pore-forming toxin-like (PFT) gene at Fhb1 was first cloned by map-based cloning and found to confer FHB resistance in wheat. Proteins often interact with each other to execute their functions. Characterization of the proteins interacting with PFT might therefore provide information on the molecular mechanisms of PFT functions. In this study, a high-quality yeast two-hybrid (Y2H) library using RNA extracted from Fusarium graminearum (Fg)-infected wheat spikes of Sumai 3 was constructed. The agglutinin domains of PFT exhibited no self-activation and toxicity to yeast cells and were used as bait to screen the Y2H library. Twenty-three proteins that interact with PFT were obtained, which were mainly involved in the ubiquitination process, clathrin coat assembly, the oxidation-reduction process, and protein phosphorylation. The expression pattern of these interacting genes was analyzed by quantitative real-time PCR. This study clarifies the protein interactions of PFT and raises a regulatory network for PFT regarding FHB resistance in wheat.