Astragaloside IV (AS-IV) is one of the main active ingredients of Astragalus membranaceus. This study is aimed to investigate AS-IV׳s effects on Ca2+ channel activity of single cardiomyocytes and single Ca2+ channels. Whole-cell Ca2+ currents in freshly dissociated cardiomyocytes were measured using the whole-cell patch-clamp technique. Single Ca2+ channel currents were examined in cell-attached patches and inside-out patches. In the whole-cell recording, AS-IV reduced the amplitude of l-type Ca2+ currents (ICaL) in a concentration-dependent manner. Although AS-IV did not alter the steady-state activation curves, the voltage dependence of the current inactivation curves was negatively shifted by AS-IV in a concentration dependent manner. Consistent with the results of the whole-cell recording, in the inside-out configuration the ensemble average of single Ba2+ current via l-type Ca2+ channel was dose-dependently reduced by AS-IV. The reduction of unitary Ba2+ current at 0.1 or 1µM AS-IV was accounted for a decrease in the channel activity (NPo). In addition to the decrease in NPo, there was a reduction of Po without a change in channel number or an apparent change in single channel current. Furthermore, we found that the open-closed kinetics of the channel were affected by AS-IV. AS-IV induced the shift of l-type Ca2+ channels from either brief openings (mode 1) or long-lasting openings (mode 2) to no active opening (mode 0). Our results suggest that AS-IV blocks the currents through Ca2+ channels in guinea-pig ventricular myocytes by affecting the open-closed kinetics of l-type Ca2+ channels to inhibit the channel activities. This study could provide theoretical basis for the drug exploiting of the monomer of Astragalus membranaceus.