The fungal pathogen Fusarium graminearum is the causal agent of fusarium head blight in wheat and other small grain cereals. This fungus is known to produce high amounts of cell wall-degrading enzymes during infection of wheat spikes. In addition, wheat tissue is particularly rich in xylan, which can be hydrolysed by fungal xylanases. In order to establish the role of F. graminearum xylanase activity in pathogenicity, targeted gene disruption of the F. graminearum xyr1 gene, encoding the major regulator of xylanase gene expression, was performed. When grown on xylan as carbon source, the xylanase activity of the Δxyr1 mutant was dramatically reduced and fungal growth was significantly reduced compared to the wildtype fungus. When grown on carboxymethylcellulose, the cellulolytic activity of the mutant was also reduced and the mutant did not grow on wheat cell walls. The disruption of the xyr1 gene greatly reduced the expression of xylanase-encoding genes both in vitro and during wheat spike infection, thus confirming the involvement of F. graminearum Xyr1 in the regulation of genes controlling xylan degradation. However, despite the deep impact caused by xyr1 gene disruption on the expression of xylanase genes and on total xylanase activity, the virulence of the Δxyr1 mutant appeared unaffected on Triticum aestivum and T. durum spikes and on soybean seedlings. In conclusion, although a possible role for residual xylanase activity in the virulence of F. graminearum cannot be conclusively excluded, the results question the importance of xylanase activity during the infection process.