In adipocytes, mitochondrial uncoupling is known to trigger a triglyceride loss comparable with the one induced by TNFα, a proinflammatory cytokine. However, the impact of a mitochondrial uncoupling on the abundance/composition of mitochondria and its connection with triglyceride content in adipocytes is largely unknown. In this work, the effects of a mild mitochondrial uncoupling triggered by FCCP were investigated on the mitochondrial population of 3T3-L1 adipocytes by both quantitative and qualitative approaches. We found that mild mitochondrial uncoupling does not stimulate mitochondrial biogenesis in adipocytes but induces an adaptive cell response characterized by quantitative modifications of mitochondrial protein content. Superoxide anion radical level was increased in mitochondria of both TNFα- and FCCP-treated adipocytes, whereas mitochondrial DNA copy number was significantly higher only in TNFα-treated cells. Subproteomic analysis revealed that the abundance of pyruvate carboxylase was reduced significantly in mitochondria of TNFα- and FCCP-treated adipocytes. Functional study showed that overexpression of this major enzyme of lipid metabolism is able to prevent the triglyceride content reduction in adipocytes exposed to mitochondrial uncoupling or TNFα. These results suggest a new mechanism by which the effects of mitochondrial uncoupling might limit triglyceride accumulation in adipocytes.