The present study aimed at analysing how dietary folic acid (FA) and Fe deficiency, followed by supplementation with these nutrients, affects the expression of folate and Fe transporters in the duodenum, as well as FA and Fe status. After a deficiency period, Wistar rats were randomised to a group fed with a diet deficient in FA and supplemented with Fe (DFE), a diet deficient in Fe and supplemented with FA, a diet supplemented with Fe and FA (FEFOL), a diet deficient in Fe and FA (D) or a control diet (C). Tissue collection was performed after 2, 10 or 21 d of these diets. Group D had higher Slc11a2 mRNA levels than the DFE group at every time point and there were differences in mRNA levels of Slc46a1 between the DFE and the FEFOL groups at the third time point, but we observed no differences in protein levels between the groups. The DFE and D groups not only had lower serum folate concentrations at every time point but also had the highest homocysteine concentrations. Total Fe binding capacity concentrations were the lowest in the DFE group at the first time point and in the DFE and the FEFOL groups at the final time point. Simultaneous supplementation with FA and Fe resulted in significantly higher Hb concentrations than did supplementation with these nutrients alone. Our findings indicate that dietary FA and Fe deficiency, and subsequent supplementation with these nutrients, affects transcription but not the protein levels of FA and Fe transporters in the duodenum.