Abstract Aim: To investigate the link between cell stiffness and volume-regulated anion current (VRAC) in aortic endothelium. Method: Bovine aortic endothelial cells (BAECs) were exposed to methyl-β-cyclodextrin (MβCD) to deplete cellular cholesterol and the changes in cellular stiffness were measured by micropipette aspiration. VRAC density was measured electrophysiologically in the same cell populations. Furthermore, to probe the effects of cholesterol depletion on the mechanics of ‘deep’ cytoskeleton, we employ a novel technique to analyse correlated motion of intracellular particles. Results: We show that cholesterol depletion results in cellular stiffening and an upregulation of VRAC density. Replenishing cellular sterol pool with epicholesterol, a chiral analogue of cholesterol, abrogates both of these effects. This indicates that cholesterol sensitivity of both cell mechanics and VRAC are due to changes in the physical properties of the membrane rather than due to specific sterol–protein interactions. We also show that cholesterol depletion increases the stiffness of the ‘deep cytoskeleton’ and that disruption of actin filaments abolishes both cell stiffening and upregulation of VRAC due to cholesterol depletion. Furthermore, comparing BAECs to human aortic endothelial cells (HAECs), we show that BAECs that are inherently stiffer also develop larger VRACs. Conclusions: Taken together, our observations suggest an increase in the cytoskeleton stiffness has a facilitatory effect on VRAC development. We suggest that stiffening of the cytoskeleton increases tension in the membrane–cytoskeleton layer and that in turn facilitates VRAC.