Objective: To evaluate the antibacterial activity and mechanism of pregnane alkaloid with novel structure (K-6) extracted from Pachysandra terminalis, and to clarify whether K-6 could be used as a candidate antibacterial agent.Methods: Susceptibility testing was performed by broth microdilution, growth curve and kill curve were evaluated by culture turbidity measurements and viable counting.The resistance induction was tested.The vivo antibacterial activity of K-6 was studied on a methicillin-resistant Staphylococcus aureus (MRSA)-infected BALB/c mice model.Mode of action studies included scanning and transmission electron microscopy(SEM and TEM).Results: K-6 showed antibacterial effects against MRSA and methicillin resistant Staphylococcus epidermidis (MRSE) with minimum inhibitory concentration (MIC) values (25 mg/L), but did not induce antibiotic resistance in bacteria.The administration of K-6 dose dependently improved the animal survival rate of mice infected with MRSA, with survival rates of 36.34% and 66.67% in the low-dose and high-dose groups, respectively.The protective effects were associated with the reduction of the bacterial titers in the blood and with the morphological amelioration of infected tissues.Scanning and transmission electron microscopy analyses indicated that the cytoplasm shrunk of LAC cells led to noticeable gaps between the cell membrane and cell cytoplasm, and the severe damaged cell membrane resulted in the leakage of intracellular content, which ultimately caused the lethal effect of K-6 on bacteria.Conclusion: These findings demonstrate that K-6 is a potential nonresistance-inducing antibiotic agent against MRSA and MRSE, which may be a leading compound for the discovery and development of new antibacterial agent.