D6 PROTEIN KINASE (D6PK) is a polarly localized plasma membrane-associated kinase from Arabidopsis thaliana that activates polarly distributed PIN-FORMED (PIN) auxin transporters. D6PK traffics rapidly to and from the plasma membrane, independently from its PIN targets. D6PK plasma membrane association is dependent on the middle domain, an insertion between kinase subdomains VII and VIII. How D6PK polar plasma membrane targeting is established and maintained remains to be understood. Here, we show that cysteines of repeated CXX(X)P motifs in the middle domain are S-acylated and required for D6PK membrane association. While D6PK S-acylation is not detectably regulated during intracellular trafficking, the phosphorylation of adjacent serine residues, in dependence of the upstream regulatory 3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASES1, regulates D6PK residence time at the plasma membrane, lateral diffusion, and trafficking. We describe a mechanism for the regulation of D6PK plasma membrane interaction and polarity, which may also explain the polarity regulation of related kinases.