Potassium (K(+)) influx into pollen tubes via K(+) transporters is essential for pollen tube growth; however, the mechanism by which K(+) transporters are regulated in pollen tubes remains unknown. Here, we report that Arabidopsis thaliana Ca(2+)-dependent protein kinase11 (CPK11) and CPK24 are involved in Ca(2+)-dependent regulation of the inward K(+) (K(+)in) channels in pollen tubes. Using patch-clamp analysis, we demonstrated that K(+)in currents of pollen tube protoplasts were inhibited by elevated [Ca(2+)]cyt. However, disruption of CPK11 or CPK24 completely impaired the Ca(2+)-dependent inhibition of K(+)in currents and enhanced pollen tube growth. Moreover, the cpk11 cpk24 double mutant exhibited similar phenotypes as the corresponding single mutants, suggesting that these two CDPKs function in the same signaling pathway. Bimolecular fluorescence complementation and coimmunoprecipitation experiments showed that CPK11 could interact with CPK24 in vivo. Furthermore, CPK11 phosphorylated the N terminus of CPK24 in vitro, suggesting that these two CDPKs work together as part of a kinase cascade. Electrophysiological assays demonstrated that the Shaker pollen K(+)in channel is the main contributor to pollen tube K(+)in currents and acts as the downstream target of the CPK11-CPK24 pathway. We conclude that CPK11 and CPK24 together mediate the Ca(2+)-dependent inhibition of K(+)in channels and participate in the regulation of pollen tube growth in Arabidopsis.