Leptin has properties of a profibrogenic cytokine. In liver the activated hepatic stellate cell (HSC) is responsible for a net production of extracellular matrix (ECM). A key molecule synthesized is the tissue inhibitor of metalloproteinase I (TIMP-1), which acts to inhibit the activity of matrix metalloproteinases (MMPs). The purpose of the present study was to determine how leptin, a gp130 cytokine, orchestrates the regulation of TIMP-1 gene activation and expression. Transient transfection of primary HSCs revealed that leptin significantly increased luciferase activity of a 229 bp TIMP-1 promoter construct (TIMP-1-229). An electrophoretic mobility shift assay (EMSA) revealed that leptin enhanced Sp1 binding. Site-directed mutagenesis for Sp1 reduced the enhancing effect of leptin on TIMP-1 transcriptional activation and this effect was dose-dependent on the number of Sp1 sites mutated. Chromatin immunoprecipitation (ChIP) revealed that leptin enhanced binding of Sp1; however, inhibition of STAT3 phosphorylation by AG490 also blocked Sp1 phosphorylation and significantly reduced leptin-associated TIMP-1-229 promoter activity, indicating that one mechanism for leptin-increased transcriptional activity is via phosphorylation of Sp1 and subsequent promoter binding. Finally, we demonstrate that leptin also results in intranuclear pSTAT3 binding to Sp1. We propose a novel mechanism whereby leptin-mediated TIMP-1 transcription employs a Sp1/pSTAT3-dependent mechanism, one of which is a non-canonical association between Sp1 and pSTAT3. These data provide a new molecular mechanism whereby the adipocytokine leptin plays a role in complications of the metabolic syndrome.