We have previously demonstrated that insulin-stimulated glucose metabolism, and not insulin per se , mediates the effects of insulin to increase the transcriptional activity of the leptin promoter in adipocytes. Here, we sought to identify the specific cis -acting DNA elements required for the upregulation of leptin gene transcription in response to insulin-mediated glucose metabolism. To accomplish this, 3T3-L1 cells and primary rat adipocytes were transfected with a series of luciferase reporter genes containing portions of the mouse leptin promoter. Using this method, we identified an element between −135 and −95 bp (relative to the transcriptional start site) that mediated transcription in response to insulin-stimulated glucose metabolism in adipocytes. This effect was abolished by incubation with 2-deoxy- d -glucose, a competitive inhibitor of glucose metabolism. Gel shift electrophoretic mobility shift assays confirmed that the stimulatory effect of insulin-mediated glucose metabolism on leptin transcription was mediated by a previously identified Sp1 site. Consistent with these findings, incubation of primary rat adipocytes with WP631, a specific inhibitor of specificity protein (Sp)1-dependent transcription, inhibited glucose- and insulin-stimulated, but not basal, leptin secretion. Together, these findings support a key role for Sp1 in the transcriptional activation of the leptin gene promoter by insulin-mediated glucose metabolism.