Cigarette smoking is known to aggravate Graves' orbitopathy (GO) severity by enhancing adipogenesis. We investigated the effect of quercetin, an antioxidant, on adipocyte differentiation induced by cigarette smoke extract (CSE) in primary cultured orbital fibroblasts (OFs) from GO patients. Freshly prepared CSE was added to the cells and H(2)O(2) was used as a positive control. Intracellular reactive oxygen species (ROS) generation and adipogenesis were measured. The expressions of proteins peroxisome proliferator-activated receptor (PPAR) γ, CCAAT-enhancer-binding proteins (C/EBP) α and β, and heme oxygenase-1 (HO-1), an antioxidant enzyme, were examined during adipogenic differentiation. In result, CSE and H(2)O(2) dose-dependently stimulated intracellular ROS production in normal and Graves' OFs. The effect of 2% CSE was similar to that of 10 μM H(2)O(2); both concentrations were noncytotoxic and were used throughout the experiment. Quercetin pretreatment reduced the ROS generation stimulated by either CSE or H(2)O(2) in preadipocyte OFs. CSE and H(2)O(2) stimulated adipocyte differentiation in cultured OFs. The addition of quercetin (50 or 100 μM) suppressed adipogenesis. Quercetin also suppressed ROS generation in differentiating OFs during adipogenesis stimulated by CSE and H(2)O(2). Additionally, the expressions of PPARγ, C/EBPα, and C/EBPβ proteins were reduced in the quercetin-treated OFs. Quercetin also reduced the CSE- and H(2)O(2)-induced upregulation of ROS and HO-1 protein in differentiated OFs and preadipocyte OFs. As shown in this study, quercetin inhibited adipogenesis by reducing ROS in vitro, supporting the use of quercetin in the treatment of GO.