High liver iron content is a risk factor for developing hepatocellular carcinoma (HCC). However, HCC cells are always iron-poor. Therefore, an association between hepatocyte iron storage capacity and differentiation is suggested. To characterize biological processes involved in iron loading capacity, we used a cDNA microarray to study the differentiation of the human HepaRG cell line, from undifferentiated proliferative cells to hepatocyte differentiated cells. We were able to identify genes modulated along HepaRG differentiation, leading us to propose new genes not previously associated with HCC. Moreover, using Gene Ontology annotations, we demonstrated that HepaRG hepatocyte iron loading capacity occurred both with the repression of genes involved in cell motility, signal transduction, and biosynthesis and with the appearance of genes linked to lipid metabolism and immune response. These results provide new insights in the understanding of the relationship between iron and hepatocyte differentiation during iron-related hepatic diseases.