AbstractL-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Ca_v1.2 and Ca_v1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Ca_v1.2 -mediated L-type calcium currents (I_CaL) at concentrations leaving Ca_v1.3-mediated I_CaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Ca_v1.2 and Ca_v1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Ca_v1.2− and Ca_v1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Ca_v1.2 Ca²⁺ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms.