Interleukin-6 (IL-6) has emerged as an important cytokine involved in the regulation of metabolism. However, the role of IL-6 in the etiology of obesity and insulin resistance is not fully understood. Mitochondria are key organelles of energy metabolism, and there is growing evidence that mitochondrial dysfunction plays a crucial role in the pathogenesis of obesity-associated insulin resistance. In this study, we determined the direct effect of IL-6 on lipolysis in adipocytes, and the effects of IL-6 on mitochondrial function were investigated. We found that cells treated with IL-6 displayed fewer lipids and an elevated glycerol release rate. Further, IL-6 treatment led to decreased mitochondrial membrane potential, decreased cellular ATP production, and increased intracellular ROS levels. The mitochondria in IL-6-treated cells became swollen and hollow with reduced or missing cristae. However, insulin-stimulated glucose transport was unaltered. PGC-1α, NRF1, and mtTFA mRNA levels were markedly increased, and the mitochondrial contents were also increased. Our results demonstrate that IL-6 can exert a direct lipolytic effect and induce mitochondrial dysfunction. However, IL-6 did not affect insulin sensitivity in adipocytes in vitro. We deduce that in these cells, enhanced mitochondrial biogenesis might play a compensatory role in glucose transport.