As essential cofactor in many proteins and redox enzymes, copper and iron are involved in a wide range of biological processes. Mild dietary deficiency of metals represents an underestimated problem for human health, because it does not cause clear signs and clinical symptoms, but it is associated to long-term deleterious effects in cardiovascular system and alterations in lipid metabolism. The aim of this work was to study the biological processes significantly affected by mild dietary deficiency of both metals in rat intestine, in order to better understand the molecular bases of the systemic metabolic alterations, as hypercholesterolemia and hypertriglyceridemia observed in copper-deficient rats. A gene-microarray differential analysis was carried out on the intestinal transcriptome of copper- and iron-deficient rats, thus highlighting the biological processes significantly modulated by the dietary restrictions. The gene array analysis showed a down-regulation of genes involved in mitochondrial and peroxisomal fatty acids beta-oxidation and an up-regulation of genes involved in plasmatic cholesterol transport (apoprotein E and lecithin:cholesterol acyltransferase) in copper deficiency. Furthermore, a severe down-regulation of ApoH was pointed out in iron-deficient animals.