Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na⁺-K⁺ pump via protein kinase (PK)C-dependent activation of NADPH oxidase. We examined whether this is mediated by oxidative modification of the pump subunits. We detected glutathionylation of ₁, but not ₁, subunits in rabbit ventricular myocytes at baseline. ₁ Subunit glutathionylation was increased by peroxynitrite (ONOO^-), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased ₁/₁ subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with ₁ subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na⁺-K⁺ pump current (I_p). Cysteine (Cys46) of the ₁ subunit was the likely candidate for glutathionylation. We expressed Na⁺-K⁺ pump ₁ subunits with wild-type or Cys46-mutated ₁ subunits in Xenopus oocytes. ONOO^- induced glutathionylation of ₁ subunit and a decrease in Na⁺-K⁺ pump turnover number. This was eliminated by mutation of Cys46. ONOO^- also induced glutathionylation of the Na⁺-K⁺ ATPase ₁ subunit from pig kidney. This was associated with a 2-fold decrease in the rate-limiting E₂E₁ conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na⁺-K⁺ pump occurs via glutathionylation of its ₁ subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na⁺ levels.