The voltage-dependent anion channel (VDAC) has emerged as a key player in mitochondrial bioenergetics. As the main pathway for calcium and mitochondrial metabolites, such as ATP and ADP, to cross the mitochondrial outer membrane, VDAC is central to maintenance of mitochondrial homeostasis. It is thus unsurprising that VDAC is implicated in nearly every mitochondria-related pathology, including cardiovascular disease, cancers, and neurodegeneration. Despite decades of research, the relatively uncomplicated structure of VDAC—a β-barrel with an internal broken α-helical belt—has hampered efforts to elucidate its functional mechanisms. Unlike other β-barrel channels, such as bacterial porins and toxins, VDAC transitions to low-conducting states, or “gates”, at relatively low voltages, less than 30 mV. In VDAC's gated state, metabolite transport is suppressed and calcium transport enhanced; thus, gating is one means by which VDAC may regulate fluxes across the mitochondrial membrane. In this talk, I will describe computational and experimental efforts that have led to identification of K12 as a key residue that is responsible for the exquisite sensitivity to voltage of its gating process [JACS 2022, 144(32) 14564-14577, DOI:10.1021/jacs.2c03316]. K12 is bistable; its motions between two widely separated positions along the pore axis enhance the fluctuations of the β-barrel and augment the likelihood of gating. These results are confirmed by single channel electrophysiology of various K12 mutants, which show a dramatic reduction of the frequency of voltage-induced gating transitions. The crystallographic structure of the K12E mutant is similar to the wild type; however, 60 µs of atomistic MD simulations using the K12E mutant structure show restricted motion of the mutated residue 12, due to enhanced connectivity with neighboring residues, and diminished amplitude of barrel motions. We conclude that β-barrel fluctuations, governed particularly by residue K12, drive VDAC gating transitions.