Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a key enzyme that plays a significant role in intracellular signaling and the modulation of mitochondrial membrane properties. It is known that the voltage-dependent anion channel (VDAC) is one of the most abundant outer mitochondrial membrane (OMM) proteins acting as a significant passageway and regulatory site for various enzymes, proteins, ions, and metabolites. Considering this, we hypothesize that VDAC could be one of the targets for CaMKII enzymatic activity. Our in vitro experiments indicate that VDAC can be phosphorylated by the CaMKII enzyme. Moreover, the bilayer electrophysiology experimental data indicate that CaMKII significantly reduces VDAC's single-channel conductivity; its open probability remains high at all the applied potentials between +60 and −60 mV, and the voltage dependency was lost, which suggests that CaMKII disrupted the VDAC's single-channel activities. Hence, we can infer that VDAC interacts with CaMKII and thus acts as a vital target for its activity. Furthermore, our findings suggest that CaMKII could play a significant role during the transport of ions and metabolites across the outer mitochondrial membrane (OMM) through VDAC and thus regulate apoptotic events.