Accumulating evidences suggest that intracellular chloride concentration ([Cl-](i)) is involved in several pathological process of cardiovascular disease. However, the role of [Cl-](i) in endothelial cells apoptosis remains unknown. Therefore, this study aims to test the function of [Cl-](i) in human umbilical vein endothelial cells (HUVECs) apoptosis induced by lipopolysaccharide (LPS). Cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry. Western blot was used to detect the mitochondria-dependent pathway. Mitochondrial membrane potential (MMP) was measured by a membrane-permeant dye JC-1. Moreover, reactive oxygen species (ROS) generation was determined by 2',7'-dichlorofluorescin diacetate (H2DCF-DA). Our results showed that LPS significantly induced a decrease of cell viability and [Cl-](i) both dose-dependently and time-dependently in HUVECs. Compared with control, treatment with LPS induced cell injury and apoptosis, concomitantly with declined Bcl-2/Bax ratio, depressed MMP, increased cytochrome c release and cleaved caspase-9 and -3 protein expressions, which were all significantly potentiated in HUVECs cultured in low Cl-medium. However, lowering [Cl-](i)-induced reactive oxygen species (ROS), cell injury and apoptosis were inhibited by mitochondrial electron transport complex inhibitor rotenone. In conclusion, decrease of [Cl-](i) promotes LPS-induced HUVECs apoptosis via increasing ROS generation derived from mitochondria, suggesting modulation of [Cl-](i) is a novel approach to prevent endothelial cells apoptosis.