BACKGROUND: Mitochondrial membrane potential (ΔΨ) depends on flux of metabolites through voltage dependent anion channels (VDAC) in the mitochondrial outer membrane. In vitro, tubulin binding induces VDAC closure. In intact cells, nocodazole (Ncz) increases free tubulin and decreases ΔΨ, whereas paclitaxel (Ptx) promotes tubulin polymerization and increases ΔΨ. These effects are attributed to closing and opening of VDAC. Here, we hypothesize that VDAC knockdown will decrease ΔΨ and block the inhibitory effect of free tubulin on ΔΨ. METHODS: HepG2 cells were transfected with individual siRNAs for VDAC1/2/3 (5 nM, Ambion) for 48 h. To assess ΔΨ, fluorescence of tetramethylrhodamine methylester (TMRM) was measured by confocal microscopy. RESULTS: VDAC1/2/3 relative mRNA abundance by qPCR was ∼39, 50 and 10%, respectively. Each siRNA decreased its corresponding VDAC mRNA by ∼90% without affecting other isoforms. After VDAC1/2/3 knockdown, TMRM fluorescence was 58, 41 and 21%, respectively, compared to 100% after non-target siRNA. In control cells, Ncz decreased TMRM fluorescence by 70%, and Ptx increased fluorescence by 51%. After knockdown of VDAC1/2/3, Ncz decreased TMRM fluorescence by 25, 22 and 9%, respectively, whereas Ptx increased fluorescence by 26, 28, and 27% . CONCLUSION: Each of the three VDAC isoforms contributes to maintenance of ΔΨ in HepG2 cells. After knockdown, the relative response to changes of free tubulin was attenuated. These results are consistent with the conclusion that free tubulin dynamically regulates ΔΨ in HepG2 cells by interacting with VDAC.