Low magnesium (Mg) has been associated with oxidative stress, an important player in aging, atherosclerosis and other vascular diseases. In vivo, low Mg and immune system activation seem to cooperate to promote endothelial dysfunction. We therefore evaluated whether exposure of human endothelial cells to low Mg in vitro determines oxidative stress features. We therefore measured intracellular reactive oxygen species (ROS) by dichlorofluorescein (DCF) fluorescence after Mg deprivation with or without treatment with H2O2 While we did not observe any alteration of DCF-detectable intracellular ROS under basal conditions, we show that, early after exposure to low Mg (2 h), endothelial cells are more sensitive to the oxidant action of H2O2 than the controls cultured in physiologic concentrations of Mg. This increase of ROS in Mg deprived cells is transient and followed by a stable reduction of DCF-fluorescence below the levels measured in the controls. We also evaluated oxidative DNA damage and observed higher 8-hydroxy-deoxyguanine levels early (2 h) after Mg deprivation in respect to the controls, both in basal conditions and after treatment with H2O2 Mg deficiency in vivo associates with the onset of an inflammatory response leading to increased circulating levels of cytokines, which trigger an oxidative response in endothelial cells. We here show that exposure to IL-1 and IL-6 significantly increased the levels of DCF-detectable ROS in cells cultured in physiologic concentrations of Mg, but not in Mg-deprived cells. We conclude that low Mg transiently leads to pro-oxidant effects. We suggest that different molecules, including pro-inflammatory cytokines, might be involved in promoting endothelial dysfunction.