Voltage-gated EAG2 channel is abundant in the brain and enhances cancer cell growth by controlling cell volume. The channel contains a cyclic nucleotide-binding homology (CNBH) domain and multiple calmodulin-binding motifs. Here we show that a raised intracellular Ca2+ concentration causes proteolytic digestion of heterologously expressed and native EAG2 channels. A treatment of EAG2-expressing cells with the Ca2+ ionophore A23187 for 1 h reduces the full-length protein by ∼80% with a concomitant appearance of 30-35-kDa peptides. Similarly, a treatment with the Ca2+-ATPase inhibitor thapsigargin for 3 h removes 30-35-kDa peptides from ∼1/3 of the channel protein. Moreover, an incubation of the isolated rat brain membrane with CaCl2 leads to the generation of fragments with similar sizes. This Ca2+-induced digestion is not seen with EAG1. Mutations in a C-terminal calmodulin-binding motif alter the degrees and positions of the cleavage. Truncated channels that mimic the digested proteins exhibit a reduced current density and altered channel gating. In particular, these shorter channels lack a rapid activation typical in EAG channels with more than 20-mV positive shifts in voltage dependence of activation. The truncation also eliminates the ability of EAG2 channel to reduce cell volume. These results suggest that a sustained increase in the intracellular Ca2+ concentration leads to proteolytic cleavage at the C-terminal cytosolic region following the CNBH domain by altering its interaction with calmodulin. The observed Ca2+-induced proteolytic cleavage of EAG2 channel may act as an adaptive response under physiological and/or pathological conditions.