The phosphoinositide-dependent kinase-1 (PDK-1) activates the serum- and glucocorticoid-inducible kinase and protein kinase B isoforms, which, in turn, are known to stimulate the renal and intestinal Na⁺-dependent glucose transporter 1. The present study has been performed to explore the role of PDK-1 in electrogenic glucose transport in small intestine and proximal renal tubules. To this end, mice expressing ∼20% of PDK-1 ( pdk1^hm) were compared with their wild-type littermates ( pdk1^wt). According to Ussing chamber experiments, electrogenic glucose transport was significantly smaller in the jejunum of pdk1^hmthan of pdk1^wtmice. Similarly, proximal tubular electrogenic glucose transport in isolated, perfused renal tubule segments was decreased in pdk1^hmcompared with pdk1^wtmice. Intraperitoneal injection of 3 g/kg body wt glucose resulted in a similar increase of plasma glucose concentration in pdk1^hmand in pdk1^wtmice but led to a higher increase of urinary glucose excretion in pdk1^hmmice. In conclusion, reduction of functional PDK-1 leads to impairment of electrogenic intestinal glucose absorption and renal glucose reabsorption. The experiments disclose a novel element of glucose transport regulation in kidney and small intestine.