Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K⁺ intake, we hypothesized that this enzyme might regulate plasma K⁺ concentration ([K⁺]). We showed in wild-type mice that renal K⁺ and TK excretion increase in parallel after a single meal, representing an acute K⁺ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK^−/−) were used to assess the impact of the enzyme on plasma [K⁺]. A single large feeding did not lead to any significant change in plasma [K⁺] in TK^+/+, whereas TK^−/− mice became hyperkalemic. We next examined the impact of TK disruption on K⁺ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK^−/− mice exhibit net transepithelial K⁺ absorption because of abnormal activation of the colonic H⁺,K⁺-ATPase in the intercalated cells. Finally, in CCDs isolated from TK^−/− mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H⁺,K⁺-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K⁺ load.