Abstract Iron (Fe) is an essential micronutrient and plays an irreplaceable role in plant growth and development. Although its uptake and translocation are important biological processes, little is known about the molecular mechanism of Fe translocation within seed. Here, we characterized a novel small kernel mutant yellow stripe like 2 (ysl2) in maize (Zea mays). ZmYSL2 was predominantly expressed in developing endosperm and was found to encode a plasma membrane-localized metal–nicotianamine (NA) transporter ZmYSL2. Analysis of transporter activity revealed ZmYSL2-mediated Fe transport from endosperm to embryo during kernel development. Dysfunction of ZmYSL2 resulted in the imbalance of Fe homeostasis and abnormality of protein accumulation and starch deposition in the kernel. Significant changes of nitric oxide accumulation, mitochondrial Fe–S cluster content, and mitochondrial morphology indicated that the proper function of mitochondria was also affected in ysl2. Collectively, our study demonstrated that ZmYSL2 had a pivotal role in mediating Fe distribution within the kernel and kernel development in maize.