Aims Recent clinical trials have proven gliflozins to be cardioprotective in diabetic and non-diabetic patients. However, the underlying mechanisms are incompletely understood. A potential inhibition of cardiac Na+/H(+)exchanger 1 (NHE1) has been suggested in animal models. We investigated the effect of empagliflozin on NHE1 activity in human atrial cardiomyocytes. Methods and results Expression of NHE1 was assessed in human atrial and ventricular tissue via western blotting. NHE activity was measured as the maximal slope of pH recovery after NH(4)(+)pulse in isolated carboxy-seminaphtarhodafluor 1 (SNARF1)-acetoxymethylester-loaded murine ventricular and human atrial cardiomyocytes. NHE1 is abundantly expressed in human atrial and ventricular tissue. Interestingly, compared with patients without heart failure (HF), atrial NHE1 expression was significantly increased in patients with HF with preserved ejection fraction and atrial fibrillation. The largest increase in atrial and ventricular NHE1 expression, however, was observed in patients with end-stage HF undergoing heart transplantation. Importantly, acute exposure to empagliflozin (1 mu mol/L, 10 min) significantly inhibited NHE activity to a similar extent in human atrial myocytes and mouse ventricular myocytes. This inhibition was also achieved by incubation with the well-described selective NHE inhibitor cariporide (10 mu mol/L, 10 min). Conclusions This is the first study systematically analysing NHE1 expression in human atrial and ventricular myocardium of HF patients. We show that empagliflozin inhibits NHE in human cardiomyocytes. The extent of NHE inhibition was comparable with cariporide and may potentially contribute to the improved outcome of patients in clinical trials.