Iron deficiency is the most common nutritional deficiency worldwide and has been linked to bone disease and impaired calcium metabolism (1,2). However, how this specifically affects the pathways of intestinal calcium absorption is unknown. Based on previous findings that paracellular calcium absorption is dominant under normal dietary calcium levels (3), we investigated the effect of diet-induced iron deficiency on paracellular calcium absorption. We focused our studies on the duodenum as this is the major segment responsible for iron absorption. Six-week-old male Sprague-Dawley rats were fed an iron deficient (ID) diet containing 2-6 ppm of iron, or a matched control (C) diet, containing 48 ppm of iron for 2-weeks. On the day of experimentation, the animals were anaesthetised with an intraperitoneal injection of pentobarbitone sodium (45mg/kg) and the effect of iron deficiency on duodenal calcium absorption was tested using the in vivo ligated loop technique. A calcium concentration of 100mM was used to favour the paracellular route. At the end of the experiment, serum and plasma samples were collected via cardiac puncture for iron, ferritin and calcium assays. Additionally, duodenal mucosa was collected for RT-qPCR and Western blotting to examine the potential proteins involved in changes in calcium absorption. Following tissue collection, the animals were killed by excising the heart. Values are presented as means ± S.E.M and analysed using an unpaired student’s t-test. A significant decrease in serum iron (C: 19.6 ± 2.3 vs ID: 8.6 ± 1.3 µmol/L p<0.01, n=4-7) and plasma ferritin (C: 172.7 ± 37.9 vs ID: 41.5 ± 10.8 ng/ml p<0.05, n=4) levels confirmed that the rats were iron deficient. Although serum calcium levels were unaffected (C: 2.03 ± 0.02 vs ID: 2.09 ± 0.04 mM, n=11-12), diet-induced iron deficiency significantly increased duodenal calcium absorption (C: 197.7 ± 14.0 vs ID: 265.3 ± 3.2 nmoles of calcium transferred in to 1ml of plasma per 5cm, p<0.05, n=5) after 30 minutes. Iron deficiency upregulated vitamin D receptor (VDR) mRNA (C: 0.0153 ± 0.0008 vs ID: 0.0229 ± 0.0016 a.u. p<0.01, n=5-6) and protein (C: 0.07 ± 0.02 vs ID: 0.7 ± 0.2 a.u. p<0.05, n=5-6) expression, but did not affect circulating vitamin D levels (C: 150.7 ± 7.8 vs ID: 144.0 ± 7.2 fmol/L n=6). Interestingly, mRNA (C: 0.0004 ± 0.0001 vs ID: 0.0035 ± 0.0007 a.u. p<0.001, n=5-6) and protein (C: 0.20 ± 0.06 vs ID: 0.67 ± 0.10 a.u. p<0.05, n=3) expression of the vitamin D-sensitive claudin 2 also increased. This is the first study demonstrating that diet-induced iron deficiency increases duodenal paracellular calcium absorption via a mechanism that may involve VDR and claudin 2. Further studies are required to elucidate the cellular mechanisms involved and how these changes relate to the altered calcium metabolism and bone disease seen in individuals with iron deficiency.