Drought is a major abiotic stress that threatens maize production globally. A previous genomewide association study identified a significant association between the natural variation of ZmTIP1 and the drought tolerance of maize seedlings. Here, we report on comprehensive genetic and functional analysis, indicating that ZmTIP1, which encodes a functional S-acyltransferase, plays a positive role in regulating the length of root hairs and the level of drought tolerance in maize. We show that enhancing ZmTIP1 expression in transgenic Arabidopsis and maize increased root hair length, as well as plant tolerance to water deficit. In contrast, ZmTIP1 transposon-insertional mutants displayed the opposite phenotype. A calciumdependent protein kinase, ZmCPK9, was identified as a substrate protein of ZmTIP1, and ZmTIP1-mediated palmitoylation of two cysteine residues facilitated the ZmCPK9 PM association. The results of this research enrich our knowledge about ZmTIP1-mediated protein S-acylation modifications in relation to the regulation of root hair elongation and drought tolerance. Additionally, the identification of a favourable allele of ZmTIP1 also provides a valuable genetic resource or selection target for the genetic improvement of maize.