The Na(+), glucose cotransporter SGLT1 (SLC5A1) accomplishes Na(+)-dependent concentrative cellular glucose uptake. SGLT1 activity is enhanced by the serum and glucocorticoid inducible kinase SGK1. As shown recently, the stimulating effect of protein kinase B on the glucose carrier GLUT4 involves the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments thus explored whether PIKfyve is similarly involved in the SGK1-dependent regulation of SLC5A1. In Xenopus oocytes expressing SLC5A1 but not in water injected oocytes glucose induced a current which was significantly enhanced by coexpression of PIKfyve. The effect of PIKfyve on SLC5A1 was blunted by additional coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase (K119N)SGK1 and mimicked by coexpression of constitutively active (S422D)SGK1. The stimulating effect of PIKfyve was abrogated by replacement of the serine in the SGK consensus sequence by alanine ((S138A)PIKfyve). Moreover, coexpression of (S138A)PIKfyve significantly blunted the effect of SGK1 on SLC5A1 activity. The observations disclose that PIKfyve participates in the SGK1-dependent regulation of SLC5A1.