Hemoglobin hydrolysate is derived from the enzymatic degradation of hemoglobin. This work aimed to evaluate whether hemoglobin hydrolysate promotes the absorption of non-heme iron and the safety of absorbed iron in mice by analyzing the iron binding content, iron circulation, and liver homeostasis. We found that hemoglobin hydrolysate promoted the absorption of non-heme iron with high efficiency in duodenum by spontaneously binding non-heme iron during digestion, and increased hepatic iron content by up-regulating divalent metal transporter 1, zinc transporter 14, but hepatic iron content only increased at 3 weeks. Duodenal iron entered the blood through ferroportin without restriction at 3 weeks, and excessive iron entered the liver and then affected the hepatocyte membranes permeability and lipid synthesis through oxidative stress. With the prolongation of dietary intervention, the up-regulated hepcidin acted on the ferroportin to restrict excess iron from entering the blood, and then the hepatic homeostasis recovered. In addition, hemoglobin hydrolysate enhanced the hepatic antioxidant capacity. Taken together, hemoglobin hydrolysate has a strong ability to promote the absorption of non-heme iron in vivo, and the absorbed iron is relatively safe due to the regulation of hepcidin.