Riboflavin (vitamin B2) derivatives serve as cofactors for a very wide variety of redox enzymes but are now also known to participate in the catalysis of certain non-redox reactions and as cofactors of blue-light photoreceptors. In parallel with the unique features of its chemical reactivity, the vitamin is biosynthesised from one molecule of GTP and two molecules of ribulose phosphate by a mechanistically unique series of enzyme-catalysed reactions. Although the work on its biosynthesis has predominantly involved microorganisms, a reasonably detailed picture is now also emerging for plants. A central topic of this review is the emerging role of riboflavin biosynthesis enzymes in connection with plant's iron acquisition and pathogen resistance.