<p>FKBP51 reportedly antagonizes insulin signaling by reducing Akt activity, thus contributing to insulin resistance and weight gain. This has been well established in <i>FKBP51</i> knock-out mice. However, the effects of FKBP51 overexpression and its associated mechanisms have not been explored in liver cells. Here, we addressed this issue with a focus on mitochondrial bioenergetics. We overexpressed FKBP51 in the hepatocarcinoma cell line HepG2 and analyzed the changes in insulin signaling and mitochondrial function. FKBP51 overexpression impaired insulin-induced Akt and FOXO1 phosphorylation; however, downstream glycogen synthesis and gluconeogenesis inhibition were exacerbated. At the mitochondrial level, both insulin and FKBP51 overexpression decreased respiration, transmembrane potential, and ATP production, which explains why FKBP51 fails to prevent insulin metabolic response. Mechanistically, reduced ER-to-mitochondria Ca²⁺ transfer explains the drop in mitochondrial bioenergetics. Altogether, our results suggest that FKBP51 has a dual role in insulin responsiveness: it promotes insulin resistance proximal to the insulin receptor but has a negative impact on mitochondrial function, resulting in an accentuated effect on carbohydrate anabolism.</p>