In addition to its function as a neurotransmitter and vascular active molecule, serotonin is also a mitogen for hepatocytes and promotes liver regeneration. A possible role in hepatocellular cancer has not yet been investigated. Human hepatocellular cancer cell lines Huh7 and HepG2 were used to assess the function of serotonin in these cell lines. Characteristics of autophagy were detected with transmission electron microscopy, immunoblots of microtubule-associated protein light chain 3(LC3) and p62 (sequestosome 1). Immunoblots of the mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4E-BP1 were used to investigate signaling pathways of serotonin. Two different animal models served as principle of proof of in vitro findings. Clinical relevance of the experimental findings was evaluated with a tissue microarray from 168 patients with hepatocellular carcinoma. Serotonin promotes tumor growth and survival in starved hepatocellular carcinoma cells. During starvation hepatocellular carcinoma cells exhibited characteristics of autophagy, which disappeared in serotonin-treated cells. Rapamycin, an inhibitor of mTOR, is known to induce autophagy. Serotonin could override rapamycin by an mTOR-independent pathway and activate common downstream signals such as p70S6K and 4E-BP1. In two tumor models of the mouse, inhibition of serotonin signaling consistently impaired tumor growth. Human biopsies revealed expression of the serotonin receptor HTR2B, correlating with downstream signals, e.g., phosphorylated p70S6K and proliferation. Conclusion: This study provides evidence that serotonin is involved in tumor growth of hepatocellular cancer by activating downstream targets of mTOR, and therefore serotonin-related pathways might represent a new treatment strategy. (HEPATOLOGY 2010.).