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Efficient micronutrient acquisition is a critical factor in selecting micronutrient dense crops for human consumption. Enhanced exudation and re-uptake of metal chelators, so-called phytosiderophores, by roots of graminaceous plants has been implicated in efficient micronutrient acquisition. We compared PS biosynthesis and exudation as a response mechanism to either Fe, Zn or Cu starvation. Two barley (Hordeum vulgare L.) lines with contrasting micronutrient grain yields were grown hydroponically and PS exudation (LC-MS) and root gene expression (RNAseq) were determined after either Fe, Zn, or Cu starvation. The response strength of the PS pathway was micronutrient dependent and decreased in the order Fe > Zn > Cu deficiency. We observed a stronger expression of PS pathway genes and greater PS exudation in the barley line with large micronutrient grain yield suggesting that a highly expressed PS pathway might be an important trait involved in high micronutrient accumulation. In addition to several metal specific transporters, we also found that the expression of IRO2 and bHLH156 transcription factors was not only induced under Fe but also under Zn and Cu deficiency. Our study delivers important insights into the role of the PS pathway in the acquisition of different micronutrients. • Efficient micronutrient acquisition is a critical factor in selecting micronutrient dense crops for human consumption. • We investigated the phytosiderophore (PS) pathway response under Fe, Zn or Cu starvation in two barley lines. • Our results demonstrate that the PS pathway is not only triggered under Fe and Zn deficiency but also under Cu starvation. • PS gene expression, PS exudation, and micronutrient uptake were generally greater in the micronutrient efficient line. • These findings suggest a potential link between a highly expressed PS pathway and high micronutrient accumulation. [ABSTRACT FROM AUTHOR]