Aspartyl-(asparagyl)-beta-hydroxylase (AAH) is overexpressed in various malignant neoplasms, including hepatocellular carcinomas (HCCs). The upstream regulation of AAH and its functional role in Notch-mediated signaling and motility in HCC cells was accessed. The mRNA transcript levels of AAH, insulin receptor substrate (IRS), insulin and insulin-like growth factor (IGF) receptors and polypeptides, Notch, Jagged, and HES were measured in 15 paired samples of HCC and adjacent HCC-free human liver biopsy specimens using real-time quantitative RT-PCR and Western blot analysis. Overexpression of AAH was detected in 87% of the HCC relative to the paired HCC-free liver tissue. IRS-1, IRS-2, and IRS-4 were each overexpressed in 80% of the HCC samples, and IGF-I and IGF-2 receptors were overexpressed in 40% and 100% of the HCCs, respectively. All HCC samples had relatively increased levels of Notch-1 and HES-1 gene expression. Overexpression of AAH led to increased levels of Notch, and co-immunoprecipitation experiments demonstrated a direct interaction between AAH and Notch as well as its ligand Jagged. In conclusion, contributions to the malignant phenotype of HCC is due to activation of IGF-I and IGF-II signaling that results in over-expression of both AAH and Notch. The functional role of AAH in relation to cell motility has been linked to increased activation of the Notch signaling pathway.