In female, inadequate iron supply is a highly prevalent problem that often leads to iron-deficiency anemia. This study aimed to understand the effects of pregnancy and lactation on iron metabolism. Rats with different days of gestation and lactation were used to determine the variations in iron stores and serum iron level and the changes in expression of iron metabolism-related proteins, including ferritin, ferroportin 1 (FPN1), ceruloplasmin (Cp), divalent metal transporter 1 (DMT1), transferrin receptor 1 (TfR1), and the major iron-regulatory molecule-hepcidin. We found that iron stores decline dramatically at late-pregnancy period, and the low iron store status persists throughout the lactation period. The significantly increased FPN1 level in small intestine facilitates digestive iron absorption, which maintains the serum iron concentration at a near-normal level to meet the increase of iron requirements. Moreover, a significant decrease of hepcidin expression is observed during late-pregnancy and early-lactation stages, suggesting the important regulatory role that hepcidin plays in iron metabolism during pregnancy and lactation. These results are fundamental to the understanding of iron homeostasis during pregnancy and lactation and may provide experimental bases for future studies to identify key molecules expressed during these special periods that regulate the expression of hepcidin, to eventually improve the iron-deficiency status.