As a mediator of insulin-regulated gene expression, the FoxO1 transcription factor represents a master regulator of liver glucose metabolism. We previously reported that the high-mobility group AT-hook 1 (HMGA1) protein, a molecular switch for the insulin receptor gene, functions also as a downstream target of the insulin receptor signaling pathway, representing a critical nuclear mediator of insulin function. Here, we investigated whether a functional relationship existed between FoxO1 and HMGA1, which might help explain insulin-mediated gene transcription in the liver. To this end, as a model study, we investigated the canonical FoxO1-HMGA1-responsive IGFBP1 gene, whose hepatic expression is regulated by insulin. By using a conventional GST-pull down assay combined with co-immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) analyses, we provide evidence of a physical interaction between FoxO1 and HMGA1. Further investigation with chromatin immunoprecipitation, confocal microscopy, and Fluorescence Recovery After Photobleaching (FRAP) technology indicated a functional significance of this interaction, in both basal and insulin-stimulated states, providing evidence that, by modulating FoxO1 transactivation, HMGA1 is essential for FoxO1-induced IGFBP1 gene expression, and thereby a critical modulator of insulin-mediated FoxO1 regulation in the liver. Collectively, our findings highlight a novel FoxO1/HMGA1-mediated mechanism by which insulin may regulate gene expression and metabolism.