Active migration of tumor cells is a prerequisite for the development of metastasis and tumor progression, and is regulated by a variety of extracellular ligands. Epidemiological studies have shown that obesity increases the risk of colon cancer by 1.5- to 2-fold with obesity-associated colon cancer accounting for 14-35% of total incidence. In obese individuals, serum levels of leptin are markedly increased, and therefore, we have investigated the impact of this adipocytokine on the migration of various human colon carcinoma cell lines such as SW480, SW620, and HCT116. Leptin significantly enhanced the migratory activity of all three cell lines, and the strongest effect was observed in SW480 cells, which increased their locomotor activity from 28% spontaneously locomoting cells to 50%. The intracellular signal transduction regulating this pro-migratory effect involves the activation of the transcription factor signal transducer and activator of transcription-3 via Janus kinases, but also the activity of src tyrosine kinases, focal adhesion kinase, exclusively protein kinase Cdelta, and the phosphatidyl-inositol-3-kinase, as proven by the use of particular inhibitors and target-specific small interfering RNAs. Herein, we deliver new evidence for a modulatory role of leptin in the regulation of colon cancer progression by stimulating tumor cell migration. Thus, our findings have potential clinical implications, because understanding the impact of leptin on tumor cell migration and the underlying signal transduction mechanisms is mandatory for future development of novel therapeutics to treat obesity-associated colorectal cancer.