Hereditary hemochromatosis (HH) is a condition in which intestinal iron absorption is greatly elevated. Present treatment is weekly phlebotomy, affecting quality of life and leading to recurrent infections. The iron transporter divalent metal transporter-1 (DMT-1) of enterocytes is responsible for iron uptake from the intestinal lumen; iron is further extruded into the blood by the basolateral transporter ferroportin-1. A therapeutic approach for HH could start with a long-term reduction of iron transport by reduction of DMT-1 levels. We designed an AAV vector coding for a short antisense RNA (AAV-DMT-1-AS) against DMT-1, which reduced iron uptake by 50-60% in human intestinal cells (Caco-2). At low infection levels, DMT-1 mRNA virtually disappeared, suggesting RNAi-like and/or RNase H antisense effects. DMT-1 mRNA levels returned to normal at higher infection levels, indicating that an additional mechanism of mRNA occupation, able to block DMT-1 translation and to avoid feedback regulation by iron responsive elements (IRE), also exists. Cell morphology was normal in all cases and no increases in the interferon-related responses, measured by (a) 2'-5' A oligo synthetase (b) IFITM1 and (c) ISGF3gamma mRNA levels, were observed. Studies presented herein indicate that enterocyte targeting with a gene coding for a short antisense against iron transport blocks enterocyte iron uptake, which may have therapeutic value.