Obesity is associated with chronic low-grade inflammation. Thus, at metabolically relevant sites, including adipose tissue and muscle, there is abnormal production of proinflammatory cytokines such as TNF-α. Here we demonstrate that eNOS expression was reduced, with a concomitant reduction of mitochondrial biogenesis and function, in white and brown adipose tissue and in the soleus muscle of 3 different animal models of obesity. The genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in fat and muscle; this was associated with less body weight gain than in obese wild-type controls. Furthermore, TNF-α downregulated eNOS expression and mitochondrial biogenesis in cultured white and brown adipocytes and muscle satellite cells of mice. The NO donors DETA-NO and SNAP prevented the reduction of mitochondrial biogenesis observed with TNF-α. Our findings demonstrate that TNF-α impairs mitochondrial biogenesis and function in different tissues of obese rodents by downregulating eNOS expression and suggest a novel pathophysiological process that sustains obesity.