Pancreatic duodenal homeobox-1 (PDX-1) is a homeodomain protein that plays an important role in the development of the pancreas and in maintaining the identity and function of the islets of Langerhans. It also regulates the expression of the insulin gene in response to changes in glucose and insulin concentrations. Glucose and insulin regulate PDX-1 by way of a signaling pathway involving phosphatidylinositol 3-kinase (PI 3-kinase) and SAPK2/p38. Activation of this pathway leads to phosphorylation of PDX-1 and its movement into the nucleus. To investigate the intracellular trafficking of PDX-1, immunocytochemistry was used to localize PDX-1 in the human β-cell line NesPDX-1, in which PDX-1 is overexpressed, and in MIN6 β-cells. In low-glucose conditions, PDX-1 localized predominantly to the nuclear periphery, with some staining in the cytoplasm. After stimulation with glucose, PDX-1 was present in the nucleoplasm. The translocation of PDX-1 to the nucleoplasm was complete within 15 min and occurred in 5−10 mmol/l glucose. Insulin and sodium arsenite, an activator of the stress-activated pathway, also stimulated PDX-1 movement from the nuclear periphery to the nucleoplasm. When cells were transferred between high glucose− and low glucose−containing medium, PDX-1 rapidly shuttled between the nuclear periphery and the nucleoplasm. Glucose- and insulin-stimulated translocation of PDX-1 to the nucleoplasm was inhibited by wortmannin and SB 203580, indicating that a pathway involving PI 3-kinase and SAPK2/p38 was involved; translocation was unaffected by PD 098959 and rapamycin, suggesting that neither mitogen-activated protein kinase nor p70^s6k were involved. Arsenite-stimulated import of PDX-1 into the nucleus was inhibited by SB 203580 but not by wortmannin. Export from the nucleoplasm to the nuclear periphery was inhibited by calyculin A and okadaic acid, suggesting that dephosphorylation of PDX-1 was involved. These results demonstrated that PDX-1 shuttles between the nuclear periphery and nucleoplasm in response to changes in glucose and insulin concentrations and that these events are dependent on PI 3-kinase, SAPK2/p38, and a nuclear phosphatase(s).