In skeletal muscle, the transcription factors Foxo1 and Foxo3A control expression of proteins that mediate muscle atrophy, making the nuclear concentration and nuclear-cytoplasmic movements of Foxo1 and Foxo3A of therapeutic interest in conditions of muscle wasting. Here, we use Foxo-GFP fusion proteins adenovirally expressed in cultured adult mouse skeletal muscle fibers to characterize the time course of nuclear efflux of Foxo1-GFP in response to activation of the insulin-like growth factor-1 (IGF-1)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway to determine the time course of nuclear influx of Foxo1-GFP during inhibition of this pathway and to show that Akt mediates the efflux of nuclear Foxo1-GFP induced by IGF-1. Localization of endogenous Foxo1 in muscle fibers, as determined via immunocytochemistry, is consistent with that of Foxo1-GFP. Inhibition of the nuclear export carrier chromosome region maintenance 1 by leptomycin B (LMB) traps Foxo1 in the nucleus and results in a relatively rapid rate of Foxo1 nuclear accumulation, consistent with a high rate of nuclear-cytoplasmic shuttling of Foxo1 under control conditions before LMB application, with near balance of unidirectional influx and efflux. Expressed Foxo3A-GFP shuttles ∼20-fold more slowly than Foxo1-GFP. Our approach allows quantitative kinetic characterization of Foxo1 and Foxo3A nuclear-cytoplasmic movements in living muscle fibers under various experimental conditions.