We investigated whether a cholic acid (CA)-supplemented diet and marginal iron deficiency (MID) diet influence hepatic lipid accumulation and iron balance in rats for 2 weeks. The CA diet enhanced hepatic lipid accumulation and modulated iron metabolism such as enhancement of fecal iron excretion, reduction in iron absorption, and no alteration in plasma iron levels. The MID diet did not alter hepatic lipid concentrations with reduced iron concentration in the liver and plasma. In combination, influence of the CA supplementation on the hepatic iron concentration was opposite between iron-sufficient and MID conditions. In the liver, the CA diet enhanced lipocalin 2 expression, whereas the MID diet enhanced transferrin receptor 1 expression and reduced hepcidin expression. This study revealed an involvement of 12-hydroxylated bile acids in regulation of hepatic iron concentration under MID condition.