Zinc (Zn) is an essential element for plant growth and development, and Zn derived from crop plants in the diet is also important for human health. Here, we report that genetic variation inHeavy Metal-ATPase 4(HMA4) controls natural variation in leaf Zn content. Investigation of the natural variation in leaf Zn content in a world-wide collection of 349Arabidopsis thalianawild collected accessions identified two accessions, Van-0 and Fab-2, which accumulate significantly lower Zn when compared with Col-0. Both quantitative trait loci (QTL) analysis and bulked segregant analysis (BSA) identifiedHMA4as a strong candidate accounting for this variation in leaf Zn concentration. Genetic complementation experiments confirmed this hypothesis. Sequence analysis revealed that a 1-bp deletion in the third exon ofHMA4from Fab-2 is responsible for the lose of function ofHMA4driving the low Zn observed in Fab-2. Unlike in Fab-2 polymorphisms in the promoter region were found to be responsible for the weak function ofHMA4in Van-0. This is supported by both an expression analysis ofHMA4in Van-0 and through a series of T-DNA insertion mutants which generate truncatedHMA4promoters in the Col-0 background. In addition, we also observed that Fab-2, Van-0 and thehma4-2null mutant in the Col-0 background show enhanced resistance to a combination of high Zn and high Cd in the growth medium, raising the possibility that variation atHMA4may play a role in environmental adaptation.