Ferutinin, isolated from the root of Ferula hermonis and proposed to be used as an antiosteoporosis phytoestrogen, has death promoting activities in a number of cancer cells. However, the effect of ferutinin on the induction of apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, remains unclear. Given that ferutinin is a small molecule that can induce apoptosis in the cancer cells by opening the mitochondrial permeability transition pores, we therefore hypothesized that the effect of ferutinin to elicit apoptosis in human RBCs devoid of mitochondria would be minimal. This study tried to determine the in vitro effect of ferutinin on the induction of apoptosis in human RBCs. Eryptosis/erythroptosis after ferutinin treatment was examined for phosphatidylserine (PS) externalization, calcein leakage, and other apoptotic feature events by flow cytometry and confocal microscopy. Contrary to our prediction, ferutinin caused eryptosis/erythroptosis in human RBCs and simultaneously increased caspase-3 activity and the cytosolic free Ca(2+) ion level ([Ca(2+)]i). Yet, Ca(2+) seems not to be the sole mediator in ferutinin-mediated eryptosis/erythroptosis because depletion of the external Ca(2+) could not eliminate the apoptotic effect from ferutinin. Subsequent replenishment of the external Ca(2+) was able to promote PS externalization, caspase-3 activation, and rise of [Ca(2+)]i. Also, ferutinin at high dose (40 μM or above) was able to permeabilize the membrane of RBC ghosts in a way similar to that of digitonin. At low dose, ferutinin activated the P- and L-type Ca(2+) channels as the ferutinin-mediated [Ca(2+)]i rise was suppressed by the P-type (ω-agatoxin IVA) and L-type (verapamil and diltiazem) Ca(2+) channel blockers. Taken together, we report here for the first time that ferutinin induces in vitro apoptosis in human RBCs. Mechanistically, eryptosis/erythroptosis is mediated by membrane permeabilization and upregulation of [Ca(2+)]i with the activation of caspase-3.