The widely expressed chloride channel ClC-2 is stimulated by the serum and glucocorticoid inducible kinase SGK1. The SGK1-dependent regulation of several carriers involves the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether SGK1-dependent regulation of ClC-2 similarly involves PIKfyve. The conductance of Xenopus oocytes is increased more than eightfold by ClC-2 expression. In ClC-2-expressing oocytes, but not in water-injected oocytes, the current was further enhanced by coexpression of either, PIKfyve or constitutively active (S422D)SGK1. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter the current in ClC-2-expressing oocytes and abrogated the stimulation of the current by PIKfyve-coexpression. The stimulating effect of PIKfyve was abolished by replacement of the serine with alanine in the SGK1 consensus sequence ((S318A)PIKfyve). Coexpression of (S318A)PIKfyve significantly blunted the stimulating effect of (S422D)SGK1 on ClC-2-activity. In conclusion, PIKfyve is a potent stimulator of ClC-2-activity and contributes to SGK1-dependent regulation of ClC-2.