Pseudomonas aeruginosa is one of the most opportunistic bacterial pathogens in human communities. Being a potential antibacterial agent, antimicrobial peptide human beta-defensin 3-carbohydrate-binding domain (hBD3-CBD) was evaluated in this study by in vitro bactericidal test, special gene expressions, hBD3-CBD effects on biofilm formation assays, swimming, twitching, and swarming activities of P. aeruginosa PA14, and hBD3-CBD effects on the antibiotic 50 % minimal inhibitory concentration (MIC50) and 90 % minimal inhibitory concentration (MIC90) against clinical P. aeruginosa isolates. The MIC against P. aeruginosa PA14 was 32 mu g/ml; hBD3-CBD showed significant bactericidal activities when the concentration reached 8 mu g/ml, and when the concentration reached 2 mu g/ml, hBD3-CBD successfully repressed the biofilm productions in P. aeruginosa PA14. hBD3-CBD could inhibit the in vitro swimming, twitching, and swarming activities of P. aeruginosa PA14. When 5 mu g/ml hBD3-CBD was combined with antibiotics, it decreased the MIC50 and MIC90 of tetracycline, rifampicin, and streptomycin against clinical P. aeruginosa isolates. As new antibacterial agents, hBD3-CBD and other AMPs might be used together with antibiotics to deal with infections in the future, especially the skin and soft tissue infections of drug-resistant P. aeruginosa.