BACKGROUND/AIMS: We measured aspartyl (asparaginyl)-beta-hydroxylase (AAH) gene expression in human hepatocelluar carcinoma and surrounding uninvolved liver at both the mRNA and protein level and examined the regulation and function of this enzyme. METHODS: Since growth of HCC is mediated by signaling through the insulin-receptor substrate, type 1 (IRS-1), we examined-if AAH is a downstream gene regulated by insulin and IGF-1 in HCC cells. In addition, IRS-1 regulation of AAH was examined in a transgenic (Tg) mouse model in which the human (h) IRS-1 gene was over-expressed in the liver, and an in vitro model in which a C-terminus truncated dominant-negative hIRS-1 cDNA (hIRS-DeltaC) was over-expressed in FOCUS HCC cells. The direct effects of AAH on motility and invasiveness were examined in AAH-transfected HepG2 cells. RESULTS: Insulin and IGF-1 stimulation increased AAH mRNA and protein expression and motility in FOCUS and Hep-G2 cells. These effects were mediated by signaling through the Erk MAPK and PI3 kinase-Akt pathways. Over-expression of hIRS-1 resulted in high levels of AAH in Tg mouse livers, while over-expression of hIRS-DeltaC reduced AAH expression, motility, and invasiveness in FOCUS cells. Finally, over-expression of AAH significantly increased motility and invasiveness in HepG2 cells, whereas siRNA inhibition of AAH expression significantly reduced directional motility in FOCUS cells. CONCLUSIONS: The results suggest that enhanced AAH gene activity is a common feature of human HCC and growth factor signaling through IRS-1 regulates AAH expression and increases motility and invasion of HCC cells. Therefore, AAH may represent an important target for regulating tumor growth in vivo.