An increase in intracellular Ca2+ due to voltage-gated Ca2+ (CaV) channel opening represents an important trigger for a number of second-messenger-mediated effects ranging from neurotransmitter release to gene activation. Ca2+ entry occurs through the principal pore-forming protein but several ancillary subunits are known to more precisely tune ion influx. Among them, the CaVβ subunits are perhaps the most important, given that they largely influence the biophysical and pharmacological properties of the channel. Notably, several functional features may be associated with specific structural regions of the CaVβ subunits emphasizing the relevance of intramolecular domains in the physiology of these proteins. In the current report, we show that CaVβ3 contains two PEST motifs and undergoes Ca2+-dependent degradation which can be prevented by the specific calpain inhibitor calpeptin. Using mutant constructs lacking the PEST motifs, we present evidence that they are necessary for the cleavage of CaVβ3 by calpain. Furthermore, the deletion of the PEST sequences did not affect the binding of CaVβ3 to the ion-conducting CaV2.2 subunit and, when expressed in human embryonic kidney-293 cells, the PEST motif-deleted CaVβ3 significantly increased whole-cell current density and retarded channel inactivation. Consistent with this observation, calpeptin treatment of human embryonic kidney-293 cells expressing wild-type CaVβ3 resulted in an increase in current amplitude. Together, these findings suggest that calpain-mediated CaVβ3 proteolysis may be an essential process for Ca2+ channel functional regulation.