Leishmania internalize hemoglobin (Hb) via a specific receptor (HbR) for their survival. To identify the Hb-binding domain of HbR, we cloned and expressed several truncated proteins of HbR and determined their ability to bind Hb. Our findings reveal that 90% of Hb-binding activity is retained in HbR 41-80 in comparison with HbR 1-471. We synthesized a 40 amino acid peptide (SSEKMKQLTMYMIHEMVEGLEGRPSTVRMLPSFVYTSDPA) corresponding to HbR 41-80 and found that it specifically binds Hb. Subsequently, we found that the HbR 41-80 peptide completely blocks Hb uptake in both promastigote and amastigote forms of Leishmania and, thereby, inhibits the growth of the parasite. These results demonstrate that HbR 41-80 is the Hb-binding domain of HbR, which might be used as a potential therapeutic agent to inhibit the growth of Leishmania.