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Acanthopanax (A.) henryi (Oliv.) Harms contain many bioactive compounds commonly used in traditional Chinese medicine. The objective of the present study was to investigate the antibacterial activity of the single constituent, Eleutheroside K (ETSK) isolated from the leaves of A. henryi (Oliv.) Harms, against methicillin‐resistant Staphylococcus (S.) aureus (MRSA). Broth microdilution assay was used to measure the minimal inhibitory concentration (MIC) and the MIC values of ETSK against eight clinical S. aureus strains were all 50 µg ml−1. At sub‐inhibitory concentrations, a synergistic effect between oxacillin (OXA) and ETSK was confirmed using checkerboard dilution assay and time‐kill curve analysis. The bacteriostatic effect became more pronounced when ETSK was used in combination with detergent (Triton X‐100) or ATPase inhibitor (N, N′‐dicyclohexylcarbodiimide). According to western blot analysis, the down‐regulated expression of Penicillin‐binding protein 2a (PBP2a) further validated that the bacterial activity was inhibited when treated with ETSK in a dose‐dependent manner. Results based on our study verified that ETSK significantly suppressed MRSA infections and emphasized the potential application of ETSK as a novel anti‐MRSA natural drug. [ABSTRACT FROM AUTHOR]