The current study explored the Na+/K+-ATPase (NKA) inhibition-independent proarrhythmic mechanisms of cardiac glycosides (CGs) which are well-known NKA inhibitors. With the cytosolic Ca2+ chelated by EGTA and BAPTA or extracellular Ca2+ replaced by Ba2+, effects of bufadienolides (bufalin (BF) and cinobufagin (CBG)) and cardenolides (ouabain (Oua) and pecilocerin A (PEA)) on the L-type calcium current (I Ca,L) were recorded in heterologous expression Cav1.2-CHO cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). BF and CBG demonstrated a concentration-dependent (0.1 to100 µM) I Ca,L inhibition (maximal ≥50%) without and with the NKA activity blocked by 10 µM Oua. BF significantly shortened the action potential duration at 1.0 µM and shortened the extracellular field potential duration at 0.01~1.0 µM. On the other hand, BF and CBG at 100 µM demonstrated a strong inhibition (≥40%) of the rapidly activating component of the delayed rectifier K+ current (I Kr) in heterologous expression HEK293 cells and prolonged the APD of the heart of day-3 Zebrafish larva with disrupted rhythmic contractions. Moreover, hESC-CMs treated with BF (10 nM) for 24 hours showed moderate yet significant prolongation in APD90. In conclusion, our data indicate that CGs particularly bufadienolides possess cytosolic [Ca2+]i- and NKA inhibition- independent proarrhythmic potential through I Ca,L and I Kr inhibitions.