Abstract: Effect of strophanthidin (Str) on intracellular calcium concentration (［Ca²⁺］_i) was investigated on isolated ventricular myocytes of guinea pig. Single ventricular myocytes were obtained by enzymatic dissociation technique. Fluorescent signal of ［Ca²⁺］_i was detected with confocal microscopy after incubation of cardiomycytes in Tyrode’s solution with Fluo3-AM. The result showed that Str increased ［Ca²⁺］_i in a concentration-dependent manner. The ventricular myocytes began to round-up into a contracture state once the peak level of ［Ca²⁺］_i was achieved in the presence of Str (10 μmol·L^-1), but remained no change in the presence of Str (1 and 100 nmol·L^-1). Tetrodotoxin (TTX), nisodipine, and high concentration of extracellular Ca²⁺ changed the response of cardiomycytes to Str (1 and 100 nmol·L^-1), but had no obvious effects on the action of Str (10 μmol·L^-1). The elevation of ［Ca²⁺］_i caused by Str at all of the detected concentrations was partially antagonized by rynodine (10 μmol·L^-1) or the removal of Ca²⁺ from Tyrode’s solution. In Na⁺, K⁺-free Tyrode’s solution, the response of cardiomycytes in ［Ca²⁺］_i elevation to Str (10 μmol·L^-1) was attenuated, while remained no change to Str (1 and 100 nmol·L^-1). TTX, nisodipine, and high concentration of extracellular Ca²⁺ changed the response of cardiomycytes to Str at all of the detected concentrations in Na⁺, K⁺-free Tyrode’s solution. The study suggests that the elevation of ［Ca²⁺］_i by Str at the low (nomomolar) concentrations is partially mediated by the extracellular calcium influx through Ca²⁺ channel or a “slip mode conductance” of TTX sensitive Na⁺ channel. While the effect of Str at high (micromolar) concentrations was mainly due to the inhibition of Na⁺, K⁺-ATPase. Directly triggering the release of intracellular Ca²⁺ from sarcoplasmic reticulum (SR) by Str may be also involved in the mechanism of ［Ca²⁺］_i elevation.