Glutathione peroxidase 3 (GPx3) accounts for the major anti-oxidant activity in the plasma. Here, we demonstrate that downregulation of GPx3 in the plasma of obese subjects is associated with adipose GPx3 dysregulation, resulted from the increase of inflammatory signals and oxidative stress. Although GPx3 was abundantly expressed in kidney, lung, and adipose tissue, we observed that GPx3 expression was reduced selectively in the adipose tissue of several obese animal models as decreasing plasma GPx3 level. Adipose GPx3 expression was greatly suppressed by pro-oxidative conditions such as high levels of TNF and hypoxia. In contrast, the anti-oxidant N-acetyl cystein (NAC) and the anti-diabetic drug rosiglitazone increased adipose GPx3 expression in obese and diabetic db/db mice. Moreover, GPx3 overexpression in adipocytes improved high glucose-induced insulin resistance and attenuated inflammatory gene expression whereas GPx3 neutralization in adipocytes promoted expression of proinflammatory genes. Taken together, these data suggest that suppression of GPx3 expression in the adipose tissue of obese subjects might constitute a vicious cycle to expand local ROS accumulation in adipose tissue potentially into systemic oxidative stress and obesity-related metabolic complications.