We investigated the lytic activity of the bacteriophage endolysin (lysin) LysKΔamidase against live methicillin-resistant and-susceptible staphylococcal strains clinically isolated from bovine milk and humans from different origins of China. Antibiotic resistance patterns, multilocus sequence typing and SCCmec type of 137 staphylococcal strains isolated from bovine milk associated with bovine mastitis and human diseases were studied. A lytic enzyme, LysKΔamidase, was constructed by fusing the N-terminal 220 amino acids with the C-terminal 105 amino acids of staphylococcal phage lysin LysK. Herein, the antimicrobial activity of LysKΔamidase against 66 methicillin-resistant staphylococcal strains and 71 methicillin-susceptible staphylococcal strains isolated from bovine milk and from humans in China were studied. Our results show that the lysin displayed a broad lytic spectrum; in vitro treatment killed all 137 of the milk and clinical isolates of staphylococci strains tested, including MRSA, methicillin-susceptible S. aureus (MSSA), MR-Staphylococcus hominis ssp. homins, MR-Staphylococcus epidermidis and MR-Staphylococcus haemolyticus as evidenced by scanning electron microscopy, transmission electron microscopy, turbidity reduction assay and disruption of biofilms. The present results suggest that LysKΔamidase has the potential to be an alternative therapeutic agent against pathogenic methicillin-resistant and-susceptible staphylococcal strains isolated from China.,Copyright © 2017 Elsevier Ltd. All rights reserved.