Background Primary 12α-hydroxylated bile acids (12αOH BAs) enhance intestinal iron uptake due to their ability ex vivo to chelate iron. However, no information is available on their role in vivo, especially in the liver. Objectives To investigate the effects and mechanisms of primary 12αOH BAs on hepatic iron concentration in vivo. Methods Male Wistar King A Hokkaido male rats (WKAH/HkmSlc) rats aged 4–5 weeks were fed a control diet or a diet with cholic acid (CA; 0.5 g/kg diet), the primary 12αOH BA, for 2 weeks (Study 1) or 13 weeks (Study 2). In Study 3, rats fed the same diets were given drinking water either alone or containing vancomycin (200 mg/L) for 6 weeks. The variables measured included food intake (Studies 1–3), bile acid profiles (Studies 1 and 3), hepatic iron concentration (Studies 1–3), fecal iron excretion (Studies 1 and 2), iron-related liver gene expression (Studies 2 and 3), and plasma iron–related factors (Studies 2 and 3). Results In Study 1, CA feed reduced the hepatic iron concentration (−16%; P = 0.005) without changing food intake or fecal iron excretion. In Study 2, we found a significant increase in the aortic plasma concentration of lipocalin 2 (LCN2; +65%; P < 0.001), an iron-trafficking protein. In Study 3, we observed no effect of vancomycin treatment on the CA-induced reduction of hepatic iron concentration (−32%; P < 0.001), accompanied by increased plasma LCN2 concentration (+72%; P = 0.003), in the CA-fed rats despite a drastic reduction in the secondary 12αOH BA concentration (−94%; P < 0.001) in the aortic plasma. Conclusions Primary 12αOH BAs reduced the hepatic iron concentration in rats. LCN2 may be responsible for the hepatic iron–lowering effect of primary 12αOH BAs by transporting iron out of the liver.