Aim: To determine whether different Na+/K+-ATPase signal transduction pathways have positive inotropic effects on normal ventricular myocytes (NC) and failing ventricular myocytes (FC), and are involved in an increase of [Ca2+]i induced by strophanthidin (Str). Methods: A guinea pig model of congestive heart failure was made by constricting descending aorta. The left ventricular myocytes were enzymatically isolated. The effects of 25 μmol/L Str with different signal-transducing inhibitors on contractility and the calcium transient of NC or FC from guinea pigs were simultaneously assessed and compared with those in the 25 μmol/L Str-only group by a video-based, motion-edge detection system. Results: Str at 1, 10, and 25 μmol/L in NC and Str at 0.1, 1, 10, and 25 μmol/L) in FC elevated the calcium transient amplitude and increased the positive inotropic effects in a concentration-dependent manner, respectively. At the same concentration, the effects of Str were more potent in FC than in NC. In FC, both the mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) signal transduction pathway of Na+/K+-ATPase were involved in the increase of the calcium transient induced by Str, but only activation of the MAPK pathway increased the calcium transient in NC. However, only the ROS pathway was involved in positive inotropic effects both in NC and FC. Conclusion: The present study suggests that Na+/K+-ATPase signaling pathways involved in the inotropic effects of Str in NC and FC are consistent, and Na+/K+-ATPase signaling pathways involved in the increase of [Ca2+]i by Str in NC and FC are different.