Peripheral benzodiazepine receptor (PBR) is a multifunctional protein mainly found on the outer mitochondrial membrane. PBR expression is increased by tumor necrosis factor‐α (TNF‐α) in endothelial cells. Adenoviral overexpression of PBR inhibits monocyte adhesion, VCAM‐1, and ICAM‐1 expression in TNF‐α‐activated endothelial cells. Rotenone, cyclosporine A, and bongkrekic acid suppress TNF‐α‐induced VCAM‐1 expression. Overexpression of PBR inhibits voltage‐dependent anion channel‐1 (VDAC‐1) expression and the silencing of PBR increases VDAC‐1 expression in endothelial cells. Moreover, TNF‐α‐induced VCAM‐1 expression is suppressed by VDAC‐1 gene silencing. PBR overexpression significantly decreases TNF‐α‐induced mitochondrial reactive oxygen species and MnSOD expression. These results suggest that PBR can inhibit endothelial activation and this action is related to the inhibition of mitochondrial ROS and/or VDAC‐1 expression in endothelial cells.