In the present study, antimicrobial activity and mode of a novel synthesized pyrrolizidine alkaloid (PA-1) were investigated. PA-1 exhibited predominantly strong antibacterial activity toward six bacteria tested with minimal inhibitory concentration (MIC) values ranging from 0.0039 to 0.025 mg ml-1. The time-kill assay indicated that PA-1 killed Escherichia coli and Staphylococcus aureus completely at 2MIC (minimum bactericidal concentration) within 8 h. Besides, PA-1-induced death rates of most sensitive strains (E. coli, 97.80% and S. aureus, 96.24%) were analyzed by flow cytometry. A combination of approaches was used to verify the membrane damage of E. coli and S. aureus. Results showed that release of 260 nm absorbing materials quickly increased after PA-1 treatment. PA-1 also rapidly promoted the uptake of crystal violet from 24.52 to 97.12% for E. coli and from 19.68 to 97.63% for S. aureus when the concentrations were changed from MIC to 4MIC. Furthermore, the cellular membrane damages were testified by the significant increase of fluorescence intensity and decrease of membrane potential. Finally, lecithin and phosphate groups were applied to search the possibly targets on the cytoplasmic membrane. Results showed that PA-1 acted on cytoplasmic membrane phospholipids and phosphate groups of S. aureus but not of E. coli. In conclusion, the novel synthesized PA-1 exerted its antibacterial activity by acting on membrane phospholipids and phosphate groups and then damaging the structures of cellular membrane, which finally led to cell death. © 2014 Japan Antibiotics Research Association.