Leptin controls feeding behavior and insulin secretion from pancreatic β-cells. Insulin stimulates the production of leptin, thereby establishing an adipoinsular axis. Earlier we identified leptin receptors on pancreatic β-cells and showed leptin-mediated inhibition of insulin secretion by activation of ATP-sensitive potassium channels. Here we examine transcriptional effects of leptin on the promoter of the rat insulin I gene in rodent β-cells. A fall in levels of preproinsulin mRNA is detected in vivo in islets of ob/ob mice 24 h after a single injection of leptin, in isolated ob/ob islets treated with leptin in vitro and in the β-cell line INS-1 on leptin exposure when preproinsulin mRNA expression is stimulated by 25 mM glucose or 10 nM glucagon-like peptide 1. Under these conditions, transcriptional activity of −410 bp of the rat insulin I promoter is inhibited by leptin, whereas transactivation of a 5′-deleted promoter (−307 bp) is not. The −307 sequence contains the known glucose-responsive control elements (E2:A3/4). Constitutive activation of ATP-sensitive potassium channels by diazoxide does not alter leptin inhibition of preproinsulin mRNA levels. Distinct protein–DNA complexes appear on the rat insulin I promoter sequences located between −307 and −410 with nuclear extracts from ob/ob islets in response to leptin, including a signal transducer and activator of transcription (STAT)5b binding site. These results indicate that leptin inhibits transcription of the preproinsulin gene by altering transcription factor binding to sequences upstream from the elements (307 bp) that confer glucose responsivity to the rat insulin I gene promoter. Thus leptin exerts inhibitory effects on both insulin secretion and insulin gene expression in pancreatic β-cells, but by different cellular mechanisms.