Acanthopanax senticosus(Rupr. et Maxim.) Harms (AS), a traditional herbal medicine, has been widely used to treat ischemic heart disease. However, the underlying cellular mechanisms of its benefits to cardiac function remain unclear. The present study examined the effects of total flavones from AS (TFAS) on L-type Ca2+ channel currents (ICa-L) using the whole cell patch-clamp technique and on intracellular calcium ([Ca2+]i) handling and cell contractility in rat ventricular myocytes with the aid of a video-based edge-detection system. Exposure to TFAS resulted in a concentration- and voltage-dependent blockade of ICa-L, with the half-maximal inhibitory concentration (IC50) of 283.12 μg/mL and the maximal inhibitory effect of 36.49 ± 1.95%. Moreover, TFAS not only increased the maximum current in the current–voltage relationship but also shifted the activation and inactivation curves of ICa-L toward the hyperpolarizing direction. Meanwhile, TFAS significantly reduced amplitudes of myocyte shortening and [Ca2+]i with an increase in the time to 10% of the peak (Tp) and a decrease in the time to 10% of the baseline (Tr). Thus, the cardioprotective effects of TFAS may be attributed mainly to the attenuation of [Ca2+]i through the direct inhibition of ICa-L in rat ventricular myocytes and consequent negative effect on myocardial contractility. Copyright © 2015 John Wiley & Sons, Ltd.