Background Selenium is a central determinant of antioxidative glutathione peroxidase 1 (GPx-1) expression and activity. The relevance of selenium supplementation on GPx-1 in coronary artery disease (CAD) needs to be established. We assessed the effect of selenium supplementation on GPx-1 in cell culture and on endothelial function in a prospective clinical trial. Methods Human coronary artery endothelial cells were incubated with 5.78 to 578 nmol/L sodium selenite, Se-methyl-selenocysteine hydrochloride, or seleno- l -methionine. Glutathione peroxidase 1 mRNA and protein expression and activity were measured. Coronary artery disease patients (n = 465) with impaired endothelial function (flow-mediated dilation FMD <8%) were randomly assigned to receive 200 or 500 μg sodium selenite daily or matching placebo during a 12-week period. We tested the effect on red blood cell GPx-1 activity and brachial artery FMD. Furthermore, differences in biomarkers of oxidative stress and inflammation were measured. Results Sodium selenite and Se-methyl-selenocysteine hydrochloride increased GPx-1 protein and activity in a dose-dependent manner ( P < .0001). The intention-to-treat groups comprised 433 CAD patients. Glutathione peroxidase 1 activity increased from 37.0 U/gHb (31.3-41.7) to 41.1 U/gHb (35.2-48.4) ( P < .0001) in the 200 μg and from 38.1 U/gHb (33.2-43.8) to 42.6 U/gHb (35.0-49.1) ( P < .0001) in the 500 μg sodium selenite group treated for 12-weeks. No relevant changes were observed for FMD or biomarkers of oxidative stress and inflammation. Conclusions Sodium selenite supplementation increases GPx-1 activity in endothelial cells and in CAD patients. Future studies have to demonstrate whether long-term CAD outcome can be improved.