Pathologic conditions associated with hyperinsulinemia, such as obesity, metabolic syndrome, and diabetes, seem to increase the risk of breast cancer. Here, we studied molecular mechanisms by which insulin activates the expression of leptin, an obesity hormone that has been shown to promote breast cancer progression in an autocrine or paracrine way. Using MDA-MB-231 breast cancer cells, we found that (a) insulin stimulated leptin mRNA and protein expression, which was associated with increased activation of the leptin gene promoter; (b) insulin increased nuclear accumulation of transcription factors hypoxia inducible factor (HIF)-1alpha and Sp1 and their loading on the leptin promoter; (c) small interfering RNA (siRNA)-mediated knockdown of either HIF-1alpha or Sp1 significantly down-regulated insulin-induced leptin mRNA and protein expression; further inhibition of leptin expression was observed under the combined HIF-1alpha and Sp1 siRNA treatment; (d) inhibition of extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol-3-OH kinase (PI-3K) pathways significantly, albeit partially, decreased insulin-dependent leptin mRNA and protein expression, which coincided with reduced association of HIF-1alpha and/or Sp1 with specific leptin promoter regions; and (e) inhibition of ERK1/2 reduced recruitment of both HIF-1alpha and Sp1 to the leptin promoter, whereas down-regulation of PI-3K influenced only HIF-1alpha binding. In summary, our data suggest that hyperinsulinemia could induce breast cancer progression through leptin-dependent mechanisms. In MDA-MB-231 cells, this process requires Sp1- and HIF-1alpha-mediated leptin gene transcription and is partially regulated by the PI-3K and ERK1/2 pathways.