Recently, the rapid emergence of microbial pathogens which are resistant to currently available antibiotics has triggered considerable interest searching for naturally occuring antimicrobial peptides (AMPs). Because AMPs from food organisms are comparatively nontoxic, a number of them are used as sources, purified in new antibiotics. Herein, an antibacterial peptide (heat-stable KPS-1) was isolated from Korean pen shell (Atrina pectinata) by the following procedures: solvent-extraction, heating, ultrafiltration, and RP-HPLC. The molecular weight of KPS-1 (4549.1 Da) was revealed by MALDI-TOF/MS analysis. Interestingly, KPS-1 inhibited in vitro growth of Gram-negative bacteria, including Escherichia coli, E. coli O157, Pseudomonas aeruginosa, Enterobacter sakazakii, and Salmonella typhimurium, at pH 5.2, rather than at pH 7.2. Its minimal inhibitory concentrations (MICs) were ranged from 20 to 80 µg/ml; however, it was not effective against human red blood cells at a concentration of 500 µg/ml. This suggests that this peptide is useful as a clinical agent for some human organs in an acidic environment.