<p>Cyclic nucleotide-gated channel 5 (CNGC5), CNGC6, and CNGC9 (CNGC5/6/9 for simplicity) control Arabidopsis root hair (RH) growth by mediating the influx of external Ca²⁺ to establish and maintain a sharp cytosolic Ca²⁺ gradient at RH tips. However, the underlying mechanisms for the regulation of CNGCs remain unknown. We report here that calcium dependent protein kinase 1 (CPK1) directly activates CNGC5/6/9 to promote Arabidopsis RH growth. The loss-of-function mutants <i>cpk1-1</i>, <i>cpk1-2</i>, <i>cngc5-1 cngc6-2 cngc9-1</i> (<i>shrh1</i>/<i>short root hair 1</i>), and <i>cpk1 shrh1</i> show similar RH phenotypes, including shorter RHs, more RH branching, and dramatically attenuated cytosolic Ca²⁺ gradients at RH tips. The main CPK1-target sites are identified as Ser20, Ser27, and Ser26 for CNGC5/6/9, respectively, and the corresponding alanine substitution mutants fail to rescue RH growth in <i>shrh1</i> and <i>cpk1-1</i>, while phospho-mimic versions restore the cytosolic Ca²⁺ gradient at RH apex and rescue the RH phenotypes in the same Arabidopsis mutants. Thus we discover the CPK1-CNGC modules essential for the Ca²⁺ signaling regulation and RH growth in Arabidopsis.</p>