This study was designed to assess the interactions of heme with peptides produced by enzyme hydrolysis of hemoglobin, and their relationship with heme iron absorption. Bovine hemoglobin was hydrolyzed by pepsin or by subtilisin, which differ in their hydrolysis processes. The hydrolysis rate ranged from 0 (native hemoglobin) to 15%. Heme solubility and heme-peptides interactions were compared to iron absorption by the Ussing chamber model, at intestinal pH (7.5). Increasing hemoglobin hydrolysis enhanced iron absorption; the highest value was reached between 8 and 11% hydrolysis, whatever the enzyme used. Comparing the products of hydrolysis of the two enzymes showed that heme iron absorption depends not only on its solubility, but relies mainly on the balance between the strength of heme-peptides and the polymerization rate of heme.