Gordon's syndrome resulting in hypertension and hyperkalemia is the consequence of loss-of-function mutations of WNK (with no lysine) kinase isoforms, which activates the serine/threonine oxidative stress kinase 1 (OSR1) and the closely-related STE20/SPS1-related proline/alanine-rich kinase (SPAK). OSR1 and SPAK participate in the regulation of renal salt excretion and blood pressure by stimulating the Na⁺/K⁺/2Cl^- cotransporter (NKCC2) and the Na⁺/Cl^- cotransporter (NCC). Here, we studied a role of OSR1 in the proximal renal tubule. Heterologus expression of OSR1 with and without the major electrogenic renal phosphate transporter Napi-IIa in Xenopus oocytes and subsequent recordings of the phosphate-induced current disclosed a stimulatory effect of OSR1 on Napi-IIa. According to immunohistochemical analysis OSR1 is expressed in murine proximal tubular epithelial cells. The in vivo significance of OSR1-sensitive phosphate transport was studied by comparing heterozygous knock-in mice carrying WNK kinase-resistant OSR1 (OSR^+/tg) to wild type mice (OSR^+/+). Furthermore BBMV (Brush border membrane vesicles) Napi-IIa protein expression was significantly lower in (OSR^+/tg) than (OSR^+/+) mice. Despite a significantly lower serum phosphate concentration the urinary phosphate excretion was significantly larger in OSR^185A/+ mice than in OSR^+/+ mice maintained on a normal diet or on a low-phosphate diet. The serum calcium concentration was not significantly different between the genotypes whereas the urinary calcium excretion tended to be lower in OSR^185A/+ mice compared to OSR^+/+ mice. Neither serum PTH nor calcitriol levels were significantly different between the genotypes. The serum FGF23 level was, however, significantly higher in OSR^185A/+ mice compared to OSR^+/+ mice. In conclusion, OSR1 is expressed in proximal tubular epithelial cells and participates in the regulation of Napi-IIa-dependent renal phosphate reabsorption.