The Na⁺,glutamate cotransporter EAAT3 accomplishes epithelial transport and cellular uptake of acidic amino acids. EAAT3 is stimulated by the serum and glucocorticoid inducible kinase SGK1. SGK1-dependent signaling includes the kinase PIKfyve (PIP5K3). The present study explored, whether PIKfyve participates in the regulation of EAAT3 activity. EAAT3 was expressed in Xenopus oocytes with or without kinases. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced a current (I_glu). Coexpression of either, SGK1 or PIKfyve, significantly enhanced I_glu in EAAT3 expressing oocytes. I_glu was significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressing EAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant ^K127NSGK1 did not significantly alter I_glu in EAAT3 expressing oocytes and completely reversed the stimulating effect of PIKfyve coexpression on I_glu. The stimulating effect of PIKfyve on I_glu was abolished by replacement of the serine by alanine in the SGK consensus sequence (^S318APIKfyve). Moreover, additional coexpression of ^S318APIKfyve significantly blunted I_glu in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations disclose that PIKfyve indeed participates in the SGK1-dependent regulation of EAAT3.