Abstract: An antimicrobial poly-β-peptide (β-amino acid polymer) was synthesized and characterized by nuclear magnetic resonance and gel permeation chromatography to give the number-average molecular weight of the polymer. The antibacterial activity of this poly-β-peptide against Gram positive bacteria, including multiple drug-resistant strains, was evaluated by its minimum inhibitory concentration (MIC). The MIC test showed that this poly-β-peptide had potent antibacterial activity (MIC value in the range of 3.13-12.5 μg/mL) against multiple strains of Gram positive bacteria, and it demonstrated the best activity toward B. subtilis with a MIC at 3.13 μg/mL. Investigation on possible antimicrobial resistance after repeated use of this antimicrobial poly-β-peptide indicated that S. aureus displayed no resistance to this poly-β-peptide. However, the MIC of norfloxacin hydrochloride (the antibiotic control) increased 124 folds in the same test, which indicated that S. aureus developed strong resistance easily against this antibiotic. The antibacterial mechanism of this poly-β-peptide against Gram-positive bacteria, especially methicillin-resistant S. aureus (MRSA), was also investigated using cell membrane depolarization assay and scanning electron microscopy. Results implied that this poly-β-peptide interacted with bacterial cell membrane and killed Gram positive bacteria by disrupting the cell membrane.