The genome of Fusarium graminearum, the causal agent of Fusarium head blight of wheat, contains two putative pectin methylesterase (PME)-encoding genes. However, when grown in liquid culture containing pectin, F. graminearum produces only a single PME, which was purified and identified. Its encoding gene, expressed during wheat spike infection, was disrupted by targeted homologous recombination. Two Δpme mutant strains lacked PME activity but were still able to grow on highly methyl-esterified pectin even though their polygalacturonase (PG) activity showed a reduced capacity to depolymerize this substrate. The enzymatic assays performed with purified F. graminearum PG and PME demonstrated an increase in PG activity in the presence of PME on highly methyl-esterified pectin. The virulence of the mutant strains was tested on Triticum aestivum and Triticum durum spikes, and a significant reduction in the percentage of symptomatic spikelets was observed between 7 and 12 days postinfection compared with wild type, demonstrating that the F. graminearum PME contributes to fungal virulence on wheat by promoting spike colonization in the initial and middle stages of infection. In contrast, transgenic wheat plants with increased levels of pectin methyl esterification did not show any increase in resistance to the Δpme mutant, indicating that the infectivity of the fungus relies only to a certain degree on pectin degradation.