We used Western blot analysis to examine the effect of dietary K intake on the expression of serine/threonine protein phosphatase in the kidney. K restriction significantly decreased the expression of catalytic subunit of protein phosphatase 2B (PP2B) but increased the expression of PP2B regulatory subunit in both rat and mouse kidney. However, K-depletion did not affect the expression of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). Treatment of M-1 cell, mouse CCD cells, or 293T cells with glucose oxidase (GO), which generates superoxide anions through glucose metabolism, mimicked the effect of Krestriction on PP2B expression and significantly decreased the expression of PP2B catalytic subunits. However, GO treatment increased the expression of regulatory subunit of PP2B and had no effect on the expression of PP1, PP2A and protein tyrosine phosphatase, PTP-1D. Moreover, deletion of gp91-containing NADPH oxidase abolished the effect of K depletion on PP2B. We also used patch-clamp technique to study the effect of inhibiting PP2B on ROMK channels in the cortical collecting duct (CCD). Application of cyclosporin A or FK506, inhibitors of PP2B, significantly decreased ROMK channels and the effect of PP2B inhibitors was abolished by blocking mitogen-activated-protein kinase (MAPK) of P38 and ERK. Furthermore, Western blot demonstrated that inhibition of PP2B with cyclosporin A or siRNA increased the phosphorylation of ERK and p38 MAPK. We conclude that K restriction suppresses the expression of PP2B catalytic subunits and that inhibition of PP2B decreases ROMK channel activity through stimulation of MAPK in the CCD.