<jats:title>Abstract</jats:title><jats:p><jats:italic>Fusarium graminearum</jats:italic> is a destructive wheat pathogen. No fully resistant cultivars are available. Knowledge concerning the molecular weapons of <jats:italic>F. graminearum</jats:italic> to achieve infection remains limited. Here, we report that deletion of the putative secondary metabolite biosynthesis gene cluster <jats:italic>fg3_54</jats:italic> compromises the pathogen’s ability to infect wheat through cell-to-cell penetration. Ectopic expression of <jats:italic>fgm4</jats:italic>, a pathway-specific bANK-like regulatory gene, activates the transcription of the <jats:italic>fg3_54</jats:italic> cluster in vitro. We identify a linear, C- terminally reduced and <jats:sc>d</jats:sc>-amino acid residue-rich octapeptide, fusaoctaxin A, as the product of the two nonribosomal peptide synthetases encoded by <jats:italic>fg3_54</jats:italic>. Chemically-synthesized fusaoctaxin A restores cell-to-cell invasiveness in <jats:italic>fg3_54</jats:italic>-deleted <jats:italic>F. graminearum</jats:italic>, and enables colonization of wheat coleoptiles by two <jats:italic>Fusarium</jats:italic> strains that lack the <jats:italic>fg3_54</jats:italic> homolog and are nonpathogenic to wheat. In conclusion, our results identify fusaoctaxin A as a virulence factor required for cell-to-cell invasion of wheat by <jats:italic>F. graminearum</jats:italic>.