<jats:title>Summary</jats:title><jats:p><jats:italic><jats:styled-content style="fixed-case">H</jats:styled-content>erbaspirillum seropedicae</jats:italic> <jats:styled-content style="fixed-case">Z</jats:styled-content>67 is a diazotrophic endophyte able to colonize the interior of many economically relevant crops such as rice, wheat, corn and sorghum. Structures of siderophores produced by bacterial endophytes have not yet been elucidated. The aim of this work was to identify and characterize the siderophores produced by this bacterium. In a screening for mutants unable to produce siderophores we found a mutant that had a transposon insertion in a non‐ribosomal peptide synthase (<jats:styled-content style="fixed-case">NRPS</jats:styled-content>) gene coding for a putative siderophore biosynthetic enzyme. The chemical structure of the siderophore was predicted using computational genomic tools. The predicted structure was confirmed by chemical analysis. We found that siderophores produced by <jats:italic><jats:styled-content style="fixed-case">H</jats:styled-content>. seropedicae</jats:italic> <jats:styled-content style="fixed-case">Z</jats:styled-content>67 are a suite of amphiphilic lipopeptides, named serobactin <jats:styled-content style="fixed-case">A</jats:styled-content>, <jats:styled-content style="fixed-case">B</jats:styled-content> and <jats:styled-content style="fixed-case">C</jats:styled-content>, which vary by the length of the fatty acid chain. We also demonstrated the biological activity of serobactins as nutritional iron sources for <jats:italic><jats:styled-content style="fixed-case">H</jats:styled-content>. seropedicae</jats:italic>. These are the first structurally described siderophores produced by endophytic bacteria.