SUMMARYThe secondary cell wall (SCW) is essential for providing mechanical strength to plants, enabling upright growth and facilitating the long‐distance transport of water and nutrients through the vascular system. In this study, we generated an EMS‐induced mutant library in B73 maize and identified two “drought‐sensitive like” mutants, rlck1‐1 and rlck1‐2, which exhibited impaired function of ZmRLCK1. This gene encodes a predicted receptor‐like cytoplasmic kinase with kinase activity. Phenotypic analysis revealed that ZmRLCK1 mutation disrupted metaxylem vessel development, likely compromising upward water transport. Additionally, the mutants displayed aberrant microtubule organization, suggesting a role for ZmRLCK1 in cytoskeletal regulation. Differential phosphoproteomic sequencing identified several microtubule‐associated proteins as potential substrates of ZmRLCK1, though the exact molecular mechanisms linking ZmRLCK1‐mediated phosphorylation to microtubule dynamics remain to be fully elucidated.