Obesity, a major public health problem around the world, is a strong risk factor for the development of type 2 diabetes, atherosclerosis, hypertension and cardiovascular diseases. Recent research suggests that increased production of reactive oxygen species (ROS) from accumulated fat in obesity leads to elevated systemic oxidative stress and contributes to the development of obesitylinked chronic disorders. The aim of the current study was to investigate changes of key enzymes associated with antioxidant response and metabolic pathways in 3T3-L1 cell, a preadipocyte cell line that undergoes differentiation into mature adipocytes. The changes in lipid accumulation, ROS production, glucose-6-phosphate dehydrogenase (G6PDH), superoxide dismutase (SOD) and catalase (CAT) during the course of differentiation were determined. The ROS production and G6PDH activity exponentially increased as differentiation progressed. However, CAT activity showed a sharp decrease until day 2, followed by a gradual increase up to day 6 and then leveled off. Given the importance of adipocyte differentiation and ROS production in oxidation-linked diseases, these data provide a biochemical rationale for further studies to identify cellular mechanisms that can counter ROS generation and modulate cellular redox environment in adipocytes.