Antimicrobial peptides (AMPs) are the key components of the host innate immune system and form the first line of host defense against invading pathogens. In the present study attempts were made to identify and characterize AMPs that are synthesized in the epididymal epithelial cells (EPECs) of rabbit, Oryctologus cuniculus. Ex-vivo induction of EPECs with TLR-9 ligand, CpG-ODN-2006 resulted an up-regulation of several AMPs. These molecules were identified in the acid extracts of pooled epididymal tissue homogenates using proteomic approach (Ultrafiltration, cation-exchange chromatography, RP-HPLC, Far Western blotting -FWB, 2D-PAGE and MALDI-TOF-MS). The pooled RP-HPLC fraction (peaks 1-5), named as rabbit epididymal secretory antimicrobial protein (RESAMP), showed activity against several pathogens. FWB, 2D-PAGE and MALDI-TOF-MS results revealed that of the ~19 proteins identified, four protein sequences (hemoglobin-α/β subunits, transthyretin and calreticulin) found to be reacted with HIV antigens (gp120, gp17, gp41, p24). One of the four sequences (VLSHHFGKEFTPQVQ) showed >90% homology with hemohlobin-β protein. A 29 mer peptide with VLSHHFGKEFTPQVQ sequence designated as rabbit epididymal hemoglobin beta (REHbβP). The synthesized peptide showed the highest Probability Based Mowse Score (PBMS) and demonstrated good bactericidal activity (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes and Candida albicans. REHbβP did not show cytotoxicity against human endocervical cells (End1/E6E7) and rabbit erythrocytes. Immunofluorescent results revealed that REHbβP localized in the stereocilia epididymis and on the acrosome of ejaculated spermatozoa of rabbit. To the best our knowledge, this is the first study describing the detection of AMPs in rabbit epididymis. In conclusion, the present study suggests that EPECs derived REHbβP may have therapeutic potential in the management of reproductive well being of human and animals.