GPR35 is an orphan G-protein coupled receptor that has been implicated in the development of cancer. GPR35 regulates the Na(+)/K(+)-ATPase's pump and signalling function. Here we show GPR35's critical role in ion flux that in turn controls cellular osmotic pressure and Na(+)-dependent transport in HepG2 and SW480 cells. GPR35 deficiency results in increased levels of intracellular Na(+), osmotic stress and changes in osmolytes leading to increased cells size and decreased glutamine import in vitro and in vivo. The GPR35-T108M risk variant, which increases risk for primary sclerosing cholangitis and inflammatory bowel disease, leads to lower intracellular Na(+) levels, and enhanced glutamine uptake. High salt diet (HSD) in wildtype mice resembles the intestinal epithelial phenotype of their Gpr35(-/-) littermates with decreased Goblet cell size and numbers. This indicates that GPR35's regulation of the Na(+)/K(+)-ATPase controls ion homeostasis, osmosis and Na(+)-dependent transporters.