Fusarium head blight (FHB), caused by Fusarium graminearum (F. graminearum), has become one of the most devastating wheat diseases, severely impacting both grain yield and quality. The resistance gene TaHis (encoding a histidine-rich calcium-binding protein), located at the major FHB resistance locus Fhb1, has been demonstrated to confer FHB resistance in wheat, although its underlying mechanism remains unclear. In this study, we screened a wheat yeast two-hybrid (Y2H) library and identified TaU11/U12-35K, a core component of the U12-type spliceosome (U11/U12 small nuclear ribonucleoprotein), as a novel interacting partner of TaHis. Their physical interaction was further confirmed by both Y2H and bimolecular fluorescence complementation assays. Barley stripe mosaic virus-induced gene silencing (BSMV-VIGS)-mediated knockdown of TaU11/U12-35K significantly enhanced FHB resistance in both resistant (Bainong 4299) and susceptible (Bainong 5819) cultivars compared to controls. Expression profiling revealed that TaU11/U12-35K was significantly downregulated upon F. graminearum infection in both cultivars, with consistently lower basal expression levels in Bainong 4299, suggesting a negative correlation between TaU11/U12-35K expression and FHB resistance. Collectively, our results demonstrate that TaU11/U12-35K physically interacts with TaHis and functions as a negative regulator of FHB resistance. This study provides new insights into the molecular mechanism of TaHis-mediated FHB resistance in wheat.