Abstract This work investigated serum selenium (Se) and glutathione peroxidase (GPx) levels in 25 Croatian subjects exposed to high levels of As from drinking water (median As level in urine: 620.74 μg/g creatinine) and 25 controls (32.98 μg/g creatinine). The exposed group had lower ( p < 0.001) median serum Se and GPx levels (Se: 82.34 μg/l vs 59.02 μg/l; GPx: 45.99 U/g hemoglobin vs 38.38 U/g hemoglobin). A subsample of 20 exposed subjects took part in a 2-month antioxidant supplementation trial which increased median GPx activity from 30.71 to 40.98 U/g hemoglobin ( p = 0.041) and reduced total urinary As median from 680.15 to 501.96 μg/g creatinine ( p = 0.051). The effect of selected catalase (−262C>T) and GPx1 (−593C>T) gene polymorphisms was also examined. The low Se status and GPx activity may heighten risk of adverse health effects, especially in genetically predisposed individuals. The outcome of antioxidant treatment indicates modulation of As metabolism and oxidative stress, relevance of which needs further research.