Bacteria have developed different strategies to sequester iron from the environment as this metal is essential for many biochemical processes involved in survival and host colonization. On the other hand, iron homeostasis must be fine-tuned in order to avoid deleterious effect due to iron excess. The Fur protein (Ferric uptake regulator) is a central mediator of iron homeostasis in many bacterial species. When bound to Fe²⁺, it functions as a transcriptional repressor of genes important for iron uptake and storage by binding to sequences known as "Fur-boxes" that are present in the promoter regions of target genes. In order to check if the Fur protein from Xylella fastidiosa recognizes typical Fur-boxes we performed electrophoretic mobility shift assays (EMSA) using recombinant Fur from X. fastidiosa and Fur-box containing promoter fragments from Escherichia coli and Pseudomonas aeruginosa. Recombinant Xf-Fur was expressed in E. coli and >95% pure His-tagged Xf-Fur was obtained by affinity chromatography. Our results showed that recombinant Xf-Fur is able to recognize Fur-boxes present in the promoter regions of fhuA from E. coli and pvdS from P. aeruginosa. We also performed EMSAs with DNA fragments from promoter regions of X. fastidiosa containing putative Fur-boxes, including that from tonB, exbB, fptA and fur gene. These data demonstrate the functionality of recombinant Xf-Fur and shed light on the role of Fur in the modulation of the iron regulon of this phytopathogen.