Cordycepssinensis is a prized traditionalChinese medicine and its major component cordycepin isfound to have anti-leukemia activities. However, its cyto-toxicity in erythrocytes was unclear. To examine the eVectof cordycepin on the induction of eryptosis (an apoptosis-like process in enucleated erythrocytes), Xow cytometricassays based on membrane integrity and asymmetry wereemployed. For comparison, analyses were performed inparallel with two other anti-leukemia agents, indirubin 3-monoxime (IDM) and As2O3. We found that at the IC50against leukemia HL-60, cordycepin elicited eryptosiswhile IDM and As2O3 showed no erythrotoxicity in mouseerythrocytes. Mechanistically, cordycepin increased the[Ca2+]i and activated -calpain protease in a dose-depen-dent manner. Yet, no caspase-3 activation was observed inthe cordycepin-treated erythrocytes. When extracellularCa2+ was depleted, both the cordycepin-induced eryptosisand -calpain cleavage were suppressed. Our study there-fore demonstrated for the Wrst time that cordycepin induceseryptosis through a calcium-dependent pathway in theabsence of mitochondria and caspase-3 activation