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GDP-mannose transporters (GMTs) have been implicated in the virulence of some important pathogenic fungi, and guanosine diphosphate (GDP) mannose transporters transport GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation, where mannose attaches to the modified protein. GMTs could be potential targets for new antifungal drugs, as disruption of any step in GDP-mannose biosynthesis can affect fungal viability, growth, or virulence. To date, the GDP-mannose transporter has been extensively studied in yeast, but its biological function in fungi, particularly <i>F. graminearum</i>, is still unclear. In this experimental study, the role of the GDP-mannose transporter in <i>F. graminearum</i> was investigated by analysing the <i>VRG4</i> gene. <i>FgGmtA</i> and <i>FgGmtB</i> were blastp-derived from their <i>Scvrg4</i> protein sequences and proved to be their functional homologues. The mutant and complementary strains of <i>FgGmtA</i>, <i>FgGmtB</i> and <i>FgGmtA&B</i> genes were generated and used to evaluate the effect of the two GMTs genes on mycelial growth, asexual reproduction, sexual reproduction, cell wall sensitivity, glyphosate synthesis and drug susceptibility. Only in the <i>FgGmtB</i> and <i>FgGmtA&B</i> mutants was the rate of mycelial growth slowed, conidium production increased, sexual reproduction impaired, cell wall sensitivity increased, glycemic content decreased, and drug sensitivity reduced. The results of the pathogenicity assessment of GMTs showed that only <i>FgGmtB</i> affects the patogenicity of <i>F. graminearum</i>. At the same time, the effect of GMTs on the ability of rhinoceros to synthesise DON toxins was investigated and the results showed that the ability of Δ<i>FgGmtB</i> and Δ<i>FgGmtA&B</i> mutants to produce the DON toxin was significantly reduced, and the expression of toxin-related genes was also reduced.