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Objective Mitochondria play pivotal roles in orchestrating signaling pathways in order to guarantee metabolic homeostasis under different stimuli. It has been demonstrated that the mito-nuclear communication is fundamental for facing physiological and/or stress-mediated cellular response through the activation of nuclear transcription factors. Here, we focused on the Forkhead box protein O1 (FoxO1) transcription factor that belongs to the FoxOs family proteins and is considered a “nutrients sensor” modulating the expression of nutrient-stress response genes. Methods In vitro and in vivo experimental systems, including 3T3-L1 white, X-9 beige and T37i brown adipocytes and different fat depots from C57BL/6 mice were used. The mitochondrial localization of FoxO1 was demonstrated by western blot analysis, confocal microscopy and chromatin immunoprecipitation assay, after sub-cellular compartment isolation. RT-qPCR analysis was used to evaluate the expression of antioxidant and mitochondrial genes after modulation of FoxO1 activity/localization. Treatment with diverse reactive oxygen species (ROS) species/sources were performed and assessed by cytofluorimetric analysis. Results We demonstrated that FoxO1 not exclusively localizes to cytosol and nucleus of adipocytes but also to mitochondria where it binds to mitochondrial DNA. We also proved that mitochondrial FoxO1 is phosphorylated upon normal feeding condition. Mitochondrial FoxO1 responds to starvation leaving mitochondrial compartment by ROS-mediated activation of the mitochondrial phosphatase PTPMT1. Indeed, FoxO1 de-phosphorylation and mito-to-nucleus shuttling was observed under starvation. Moreover, we provided evidence that ROS species/sources are able to differently modulate the mitochondrial localization of FoxO1. Conclusion The ability to localize at different cell compartments, including mitochondria, highlights a different layer of regulation of FoxO1 necessary for assuring a fast and efficient nutrient-stress response in white/beige adipose tissue. FoxO1 could be thus endorsed in the list of transcription factors involved in the mito-nuclear communication where ROS can act as upstream signals.