Iron (Fe) metabolism is modified by many nutritional factors. Amino acids (AA) play a central role in various biological processes, such as protein synthesis and energy supply. However, the influence of AA status on iron metabolism has not been investigated. Here, we test whether AA alters iron metabolism in an intestinal cell model. Both Fe uptake and transfer across the cell monolayer were significantly increased by non-essential AA deficiency (both p < 0.001) while only Fe transfer was increased by essential AA deficiency (p < 0.0001). Both essential and non-essential AA deficiency decreased DMT1 (±IRE) exon1A mRNA expression (respectively p = 0.0007 and p = 0.006) and increased expression of ferritin heavy chain. DMT1 + IRE (also expressing exon1A or 1B) mRNA levels were decreased by essential AA deficiency (p = 0.012). The mRNA levels of total DMT1 were also decreased by essential, but not non-essential, AA deficiency (p = 0.006). Hepcidin levels were increased significantly by non-essential amino acid deprivation (p = 0.047). Protein levels of ferroportin and/or ferritin heavy chain were not altered by AA deficiency, suggesting that they had no effect on Fe efflux or storage in the cell, though iron content of ferritin could be increased. Our data demonstrate, for the first time, that AA status affects iron transport and the expression of genes related to iron metabolism in Caco-2 cells, although the changes observed are not sufficient to explain the alteration in iron transport. We hypothesise that the effect on Fe transfer is mediated through an increased movement across the cell layer, rather than transfer across the cell membranes.