During infection, soilborne fungal pathogens face limiting conditions of different metal ions, including zinc. The role of zinc homeostasis in fungal pathogenicity on plants remains poorly understood. Here it is shown that the transcription factor ZafA, orthologous to Saccharomyces cerevisiae Zap1, functions as a key regulator of zinc homeostasis and virulence in Fusarium oxysporum, a cross-kingdom pathogen that causes vascular wilt on more than 100 plant species and opportunistic infections in humans. Expression of zafA is induced under zinc-limiting conditions and repressed by zinc. Interestingly, zafA is markedly up-regulated during early stages of plant infection, suggesting that F. oxysporum must cope with limited availability of zinc. Deletion of zafA results in deactivation of high-affinity zinc transporters, leading to impaired growth under zinc deficiency. Fusarium oxysporum strains lacking ZafA are reduced in their capability to invade and kill tomato plants and the non-vertebrate animal model Galleria mellonella. Collectively, the results indicate that ZafA-mediated adaptation to zinc deficiency is required for full virulence of F. oxysporum on plant and animal hosts. Rep, M. (2010) Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature, 464, 367-373. Maret, W. (2013) Zinc biochemistry: from a single zinc enzyme to a key element of life. Adv. Nutr. 4, 82-91. Moreno, M.A., Ibrahim-Granet, O., Vicentefranqueira, R., Amich, J., Ave, P., Leal, F., Latgé, J.-P. and Calera, J.A. (2007) The regulation of zinc homeostasis by the ZafA transcriptional activator is essential for Aspergillus fumigatus virulence. Mol. Microbiol. 64, 1182-1197.