Prohibitin (PHB‐1) is a highly conserved protein involved in mitochondrial biogenesis and function. It is secreted in lipid droplets from adipocytes and is present in the circulation. In adipose tissue it functions as a membrane receptor and can target binding partners to the mitochondria. Here we report that PHB‐1 has a hitherto undescribed role as an inhibitor of pyruvate carboxylase (PC). As a consequence, it can modulate insulin‐stimulated glucose and fatty acid oxidation. It had no effect on insulin‐stimulated 2‐deoxglucose uptake by isolated adipocytes but inhibited insulin‐stimulated oxidation of [¹⁴C]glucose with a half‐maximal concentration of ≈ 4 nm. It also inhibited oleic acid oxidation in glucose‐depleted adipocytes via depletion of oxaloacetate. In vitro experiments using broken‐cell assays confirmed that PHB‐1 inhibited PC. MALDI‐TOF analysis of proteins identified by cross‐linking of PHB‐1 to adipocyte membranes indicated that PHB‐1 is closely associated with PC and EH domain 2 (EHD2). On the basis of these data, we propose that PHB‐1 is recycled between the extracellular space and the mitochondria by a mechanism involving lipid rafts and EHD2 and can modulate mitochondrial fuel metabolism by inhibition of PC.