Neurotoxicity following South American Crotalus rattlesnake bite is primarily caused by crotoxin, the most abundant component in their venom. Despite the central role of volt-age-gated calcium channels (CaV) in neurotransmission, direct targetability by crotoxin has been poorly explored. Crotoxin is a non-covalent heterodimer formed by an acidic subunit (CA) and a basic toxic phospholipase A2 subunit (CB). Here, we chromatograph-ically isolated the CB subunit from Crotalus vegrandis and studied its effect on CaV heter-ologously expressed in HEK293 cells using whole-cell patch-clamp technique. Mass spectrometry analysis identified a protein that matched with 97% sequence coverage the CBc isoform from Crotalus durissus terrificus. Isolated CB exhibited moderate phospholipase activity that was not correlated to its cytotoxic effect on cultured tsA201 cells. Using Ba2+ as charge carrier to prevent the enzymatic activity, we found that CB inhibited currents me-diated by the N-type CaV2.2 and CaV1.2 L-type calcium channels, in a dose-dependent manner, with higher potency for the latter, and negligible changes in the voltage depend-ence of channel activation. Our results reveal a novel phospholipase-independent biolog-ical activity and a novel molecular target of CB providing new insights into the patho-physiology of Crotalus snakebite envenoming with potential clinical therapeutic implica-tions