Although the study in iron nutrition has a long history, studies on dose‐effect relationship between dietary iron and hematological parameters are still limited. The aim of this study is to clarify this relationship in rats. Fifty‐four three‐week‐old male Wistar rats were equally and randomly divided into nine groups, and fed for five weeks on graded levels of supplemental dietary ferric citrate: 0, 7, 14, 21, 28, 35 (AIN‐93G formulation), 49, 126, and 252 mg iron/kg. Hematological parameters using blood samples after overnight food withdrawal were measured. Data were analyzed by the Williams or Shirley‐Williams multiple comparison test (if significant heterogeneity of variance existed). Values between dietary iron levels 126 mg/kg and 252 mg/kg were tested by Student's t test. P value less than 0.05 was considered significant. Compared with dietary iron level 126 mg/kg where hemoglobin was highest, hemoglobin, hematocrit, MCV, and MCH were significantly lower at dietary iron level 49 mg/kg and less. Compared with dietary iron level 126 mg/kg, red blood cell counts were significantly lower at dietary iron level 7 mg/kg and less. Compared with dietary iron level 126 mg/kg, reticulocyte counts were significantly increased at dietary iron level 14 mg/kg and less. There were no significant differences in all parameters between dietary iron levels 126 mg/ kg and 252 mg/kg. These results suggest that the breakpoint of dietary iron level for hemoglobin and red cell indices is placed between 49 mg/kg and 126 mg/kg.Support or Funding InformationThis work was supported by Grants‐in‐Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI: no. 25350137).