Antimicrobial peptides/proteins (AMPs) are widely distributed in nature with vast diversity. Most AMPs share a common feature of an amphipathic structure where clusters of hydrophobic and cationic amino acids are spatially organized into discrete sectors. Ribonucleases (RNases) are abundant in living organisms and play important roles in RNA metabolism, angiogenesis, neurotoxicity, antitumor and antimicrobial activities, among which antimicrobial RNases possess high positively charged residues. To investigate the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activities against bacteria and yeast, nuclear magnetic resonance (NMR) spectroscopy and site-directed mutagenesis have been carried out. It is found that 22 positively charged residues (18 Lys and 4 Arg) of hRNase 7 exposed to the surface can be classified into three clusters, and the first cluster containing Lys1, Lys3, Lys111, Lys112 located at a flexible coil near the N terminus, rather than the catalytic residues His15, Lys38, and His123 or other two clusters, Lys32, Lys35 and Lys96, Arg97, Lys100, is critical. For most Gram-negative bacteria, the cell surface lipopolysaccharide (LPS) serves as the major target for AMPs. However, the antimicrobial activities of hRNase 7 and α-helical cationic AMPs against P. aeruginosa, a lethal pathogen to immune-compromised hospitalized patients with low antibiotic susceptibility, can be inhibited by the addition of exogenous OprI (outer membrane protein I) or anti-OprI antibody. The modification and internalization of OprI into cytosol triggered by hRNase 7 make bacterial membrane permeable to intracellular components of P. aeruginosa. Our findings highlight a novel mechanism of antimicrobial activity. This is the first report demonstrating a previously unexplored cell surface target of α-helical cationic AMPs rather than LPS, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.