Using specific inhibitors, kinase-negative mutants, and small interfering RNA against protein kinase Cα (PKCα) or PKCβI, we find that PKCβI positively regulates degranulation in rat basophilic leukemia-2H3 cells, whereas PKCα negatively regulates degranulation. Mass spectrometric and mutagenic analyses reveal that PKCα phosphorylates cofilin at Ser-23 and/or Ser-24 during degranulation. Overexpression of a nonphosphorylatable form (S23,24A), but not that of a mutant-mimicking phosphorylated form (S23,24E), increases degranulation. Furthermore, the S23,24A mutant binds to F-actin and retains its depolymerizing and/or cleavage activity; conversely, the S23,24E mutant is unable to sever actin filaments, resulting in F-actin polymerization. In addition, the S23,24E mutant preferentially binds to the 14-3-3ζ protein. Fluorescence-activated cell sorting analysis with fluorescein isothiocyanate-phalloidin and simultaneous observation of degranulation, PKC translocation, and actin polymerization reveals that during degranulation, actin polymerization is dependent on PKCα activity. These results indicate that a novel PKCα-mediated phosphorylation event regulates cofilin by inhibiting its ability to depolymerize F-actin and bind to 14-3-3ζ, thereby promoting F-actin polymerization, which is necessary for cessation of degranulation.