The Arabidopsis Yellow Stripe 1-Like (YSL) proteins have been identified by homology with the maize (Zea mays) Yellow Stripe 1 (YS1) transporter which is responsible for iron–phytosiderophore (PS) uptake by roots in response to iron shortage. Although dicotyledonous plants do not synthesize PS, they do synthesize the PS precursor nicotianamine, a strong metal chelator essential for maintenance of iron homeostasis and copper translocation. Furthermore, ZmYS1 and the rice (Oryza sativa) protein OsYSL2 have metal-nicotianamine transport activities in heterologous expression systems. In this work, we have characterized the function of AtYSL1 in planta. Two insertional loss-of-function ysl1 mutants of Arabidopsis were found to exhibit increased nicotianamine accumulation in shoots. More importantly, seeds of both ysl1 knockouts contained less iron and nicotianamine than wild-type seeds, even when produced by plants grown in the presence of an excess of iron. This phenotype could be reverted by expressing the wild-type AtYSL1 gene in ysl1 plants. ysl1 seeds germinated slowly, but this defect was rescued by an iron supply. AtYSL1 was expressed in the xylem parenchyma of leaves, where it was upregulated in response to iron excess, as well as in pollen and in young silique parts. This pattern is consistent with long-distance circulation of iron and nicotianamine and their delivery to the seed. Taken together, our work provides strong physiological evidence that iron and nicotianamine levels in seeds rely in part on AtYSL1 function.