a-hemolysin from Escherichia coli (HlyA) readily lyse erythrocytes from various species. Recently, we discovered that profound cell shrinkage and crenation precede HlyA-induced hemolysis. The present study documents the underlying mechanism for this severe volume reduction. We show that HlyA-induced shrinkage of human erythrocytes occurs subsequent to a significant rise in [Ca²⁺] _i. The following volume reduction results from stimulation of Ca²⁺-activated K⁺ channels KCa_3.1 (Gardos), as both clotrimazole and TRAM-34 prevent shrinkage and potentiate hemolysis caused by HlyA. Notably, we also found TMEM16A, a Ca²⁺-activated Cl^- channel, to be important for HlyA-induced cell shrinkage. In TMEM16A^-/- murine erythrocytes the HlyA-induced shrinkage was significantly attenuated, while lysis was higher compared with controls. Additionally, we found that HlyA leads to phosphatidyl serine (PS) exposure, which was counteracted by KCa_3.1 channel blockers. In conclusion, HlyA triggers acute erythrocyte shrinkage, which requires [Ca²⁺] _i-dependent KCa_3.1 and TMEM16A activation with subsequent PS exposure. This mechanism potentially triggers removal of HlyA- damaged erythrocytes from the blood stream, and thereby reduce the risk of intravascular hemolysis.