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Previously, we showed that the Arabidopsis active demethylase ROS1 prunes DNA methylation at the promoters of a subset of immune-responsive genes to facilitate their transcriptional activation during antibacterial defence . In particular, ROS1 was shown to demethylate the RLP43 promoter in a region carrying a functional W-box cis-element, thereby ensuring a tight binding of WRKY transcriptional factors (TFs) onto DNA. Here, we first extend these findings by showing that DNA methylation at W-box elements decreases the binding of several Arabidopsis WRKY TFs in vitro. Furthermore, we provide evidence that DNA methylation at a single cytosine located in the W-box of the RLP43 promoter strongly repels DNA binding of an Arabidopsis WRKY TF in vitro. Using structural modelling, we demonstrate that this cytosine interacts through van der Waals contacts with the conserved tyrosine of WRKY DNA binding domains. Significantly, our model predicts steric hindrance when a 5-methyl group is present on this specific cytosine, thereby likely preventing tight binding of WRKY DNA binding domains. Finally, because the WRKY motif and the residues involved in DNA contacts are conserved, we propose that this methylation-dependent WRKY-DNA binding inhibitory mechanism must be widespread across plant species.