To investigate the effects of dietary manganese (Mn) supplementation on iron (Fe) metabolism, a total of 480 50-week-old hens were fed the basal diet (control, 24.35 mg Mn/kg) without Mn supplementation for 6 weeks to reduce Mn storage in the body. Hens were then randomly assigned to one of three treatments, which included the control and control added with 60 or 300 mg Mn/kg diet (M-Mn or H-Mn). Duodenum, heart, liver, and tibia were collected in hens after 12-week feeding period. No significant differences were observed in egg production, feed/egg ratio, shell breaking strength, and shell thickness among different treatments. Compared with control or M-Mn, H-Mn decreased (P < 0.05) serum Fe concentration, while increased (P < 0.05) total Fe-binding capacity (TIBC). The Fe concentration decreased (P < 0.05) in duodenum, and tended to reduce (P < 0.10) in liver from control to M-Mn and to H-Mn; whereas, dietary Mn supplementation did not influence (P > 0.10) Fe concentration in the heart and tibia. In conjunction with reduced Fe retention, DMT1 mRNA expression decreased (P < 0.05) with dietary Mn concentration increasing in the duodenum and liver. Duodenal FPN1 mRNA level was higher (P < 0.05) in H-Mn group than that in control or M-Mn group, while hepatic FPN1 mRNA expression was lower (P < 0.05) in M-Mn or H-Mn group when compared with control. The results demonstrated that dietary Mn supplementation decreased Fe concentration in duodenum and liver of hens, which may be related to the alteration of DMT1 and FPN1 expression in these tissues.