One of the mechanisms whereby glucose stimulates insulin gene transcription in pancreatic β-cells involves activation of the homeodomain transcription factor PDX1 (pancreatic/duodenal homeobox-1) via a stress-activated pathway involving stress-activated protein kinase 2 (SAPK2, also termed RK/p38, CSBP, and Mxi2). In the present study we show, by Western blotting and electrophoretic mobility shift assay, that in human islets of Langerhans incubated in low glucose (3 mm) PDX1 exists as an inactive 31-kDa protein localized exclusively in the cytoplasm. Transfer of the islets to high (16 mm) glucose results in rapid (within 10 min) conversion of PDX1 to an active 46-kDa form that was present predominantly in the nucleus. Activation of PDX1 appears to involve phosphorylation, as shown by incorporation of32Pi into the 46-kDa form of the protein. These effects of glucose could be mimicked by chemical stress (sodium arsenite), or by overexpression of SAPK2 in the β-cell line MIN6. Overexpression of SAPK2 also stimulated PDX1-dependent transcription of a –50 to –250 region of the human insulin gene promoter linked to a firefly luciferase reporter gene. The effects of glucose were inhibited by the SAPK2 inhibitor SB 203580, and by wortmannin and LY 294002, which inhibit phosphatidylinositol 3-kinase, although the effects of stress (arsenite) were inhibited only by SB 203580. These results demonstrate that glucose regulates the insulin gene promoter through activation and nuclear translocation of PDX1 via the SAPK2 pathway.