Human β‐defensin‐3 (HBD‐3) possesses antimicrobial activities and the potential to induce proinflammatory cytokines. HBD‐3 contains a unique motif of two arginine residues (Arg or R) in the COOH‐terminal region. To understand the bioactive properties of the Arg residues of HBD‐3, we examined antimicrobial activities against Staphylococcus aureus and Pseudomonas aeruginosa using synthetic HBD‐2, HBD‐3 and two variant peptides of HBD‐3: the Arg‐truncated variant designated desR HBD‐3 and NRR HBD‐3, in which both Arg residues were shifted to the N‐terminal region. IL‐6 production from keratinocytes was studied using the peptides. HBD‐3 possessed approximately five‐fold more potent antimicrobial activities, evaluated as the minimum inhibitory concentration (MIC), against S. aureus compared with desR and NRR HBD‐3, while no significant activity was observed in HBD‐2. The antimicrobial activity of HBD‐3 against S. aureus was well preserved even at high sodium chloride concentrations, but was attenuated in desR and NRR HBD‐3. All the peptides exhibited similar antimicrobial activities against P. aeruginosa, but HBD‐2 and desR HBD‐3 showed diminished antimicrobial activities against P. aeruginosa at high salt concentrations. IL‐6 production was significantly induced in keratinocytes with HBD‐3, but not remarkably with stimulation by other peptide. These Arg residues are essential for the antimicrobial and biological properties of HBD‐3.