We demonstrated in an in vitro model (human HepG2 liver cells) that chronic hypoxia induced gene expression is associated with an aggressive phenotype in patients with hepatocellular carcinoma (HCC). The aim of this study was to characterize this model further using gene expression microarray, real-time PCR and immunocytochemistry. Subsequently, pathway analysis software was used to identify relevant processes. After examination, we selected 2% O(2) during 72h as conditions to study chronic hypoxia. The most affected signaling is centered on TGF-β1 and PPARα/RXRα. Cells at 2% O(2) showed a shift in expression of Epithelial-to-Mesenchymal-Transition (EMT) related genes. Furthermore, a downregulation of liver specific detoxification pathways including cytochrome P450's and glutathione-S-transferases was observed. Both up- and downregulation events within different signaling cascades indicated a cellular adaptation and the onset of a new equilibrium. The prominent role of TGF-β1- and PPARα/RXRα signaling and cell motility pathways warrants their further investigation for therapeutic targets in HCC.