The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK is a powerful regulator of ion transport. The study explored whether SPAK similarly regulates nutrient transporters, such as the Na(+)-coupled glucose transporter SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active (T233E)SPAK, WNK-insensitive (T233A)SPAK or catalytically inactive (D212A)SPAK, and electrogenic glucose transport determined by dual-electrode voltage-clamp experiments. Moreover, Ussing chamber was employed to determine the electrogenic glucose transport in intestine from wild-type mice (spak(wt/wt)) and from gene-targeted mice carrying WNK-insensitive SPAK (spak(tg/tg)). In SGLT1-expressing oocytes, but not in water-injected oocytes, the glucose-dependent current (I(g)) was significantly decreased following coexpression of wild-type SPAK and (T233E)SPAK, but not by coexpression of (T233A)SPAK or (D212A)SPAK. Kinetic analysis revealed that SPAK decreased maximal I(g) without significantly modifying the glucose concentration required for halfmaximal I(g) (K(m)). According to the chemiluminescence experiments, wild-type SPAK but not (D212A)SPAK decreased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I(g), which was similar in the absence and presence of SPAK, suggesting that SPAK did not accelerate the retrieval of SGLT1 protein from the cell membrane but rather down-regulated carrier insertion into the cell membrane. Intestinal electrogenic glucose transport was significantly lower in spak(wt/wt) than in spak(tg/tg) mice. In conclusion, SPAK is a powerful negative regulator of SGLT1 protein abundance in the cell membrane and thus of electrogenic glucose transport.