Fusarium graminearum is a destructive wheat pathogen. No fully resistant cultivars are available. Knowledge concerning the molecular weapons of F. graminearum to achieve infection remains limited. Here, we report that deletion of the putative secondary metabolite biosynthesis gene cluster fg3_54 compromises the pathogen's ability to infect wheat through cell-to-cell penetration. Ectopic expression of fgm4, a pathway-specific bANK-like regulatory gene, activates the transcription of the fg3_54 cluster in vitro. We identify a linear, C- terminally reduced and D-amino acid residue-rich octapeptide, fusaoctaxin A, as the product of the two nonribosomal peptide synthetases encoded by fg3_54. Chemically-synthesized fusaoctaxin A restores cell-to-cell invasiveness in fg3_54-deleted F. graminearum, and enables colonization of wheat coleoptiles by two Fusarium strains that lack the fg3_54 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 F. graminearum.