Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot (FCR) in wheat and poses a significant threat to wheat production in terms of grain yield and quality.  However, the mechanism by which F. pseudograminearum infects wheat remains unclear.  In this study, we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F. pseudograminearum.  By screening this mutant library, we identified nine independent mutants that displayed impaired pathogenesis in barley leaves.  Among these mutants, one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1, encoding essential component of the Rpd3S histone deacetylase complex in F. pseudograminearum.  To further investigate the role of FpRCO1 in F. pseudograminearum, we employed a split-marker approach to knock out FpRCO1 in F. pseudograminearum WZ-8A.  FpRCO1 deletion mutants exhibit reduced vegetative growth, conidium production, and virulence in wheat coleoptiles and barley leaves, whereas the complementary strain restores these phenotypes.  Moreover, under stress conditions, the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl, sorbitol, and SDS, but possessed reduced sensitivity to H₂O₂ compared to these characteristics in the wild-type strain.  RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression (particularly the downregulation of TRI gene expression), thus resulting in significantly reduced deoxynivalenol (DON) production.  In summary, our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development, asexual reproduction, DON production, and pathogenicity of F. pseudograminearum.  This study provides valuable insights into the molecular mechanisms underlying F. pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.