[Background] Methicillin resistant Staphylococcus aureus (MRSA) is a multidrug-resistant zoonotic pathogen that often causes many diseases such as cow mastitis. Bacterial biofilm-formation is one of the important drug-resistant mechanisms of MRSA. It has been found that the polysaccharide intercellular adhesion (PIA) regulated by ica operon, which mediates the biofilm-formation by promoting the adhesion and aggregation of MRSA, and the co-expression of the icaA and icaD genes can significantly increase the activity of N-acetylglucan transferase in S. aureus. However, it is unclear that the effect of the icaA/D protein on the biofilm-formation and drug-resistance of MRSA. [Objective] To study the relationship among icaA/D gene, biofilm-formation and drug-resistance of MRSA and provide a scientific basis for finding new drug targets. [Methods] MRSA M5 isolated from mastitis milk, which exhibited a multidrug-resistant and strong biofilm-forming ability, was used to construct the icaA/D genes deletion strain by homologous recombination technology. The biofilm-forming ability and process of the MRSA wild strain and icaA/D genes deletion strain were assessed by FITC-ConA staining combined with laser confocal microscopy. Finally, the minimum inhibitory concentrations (MICs) of 14 antimicrobial agents to wild strain and icaA/D genes deletion strain were detected by broth microdilution method. [Results] The icaA/D genes deletion strain was successfully constructed. Through observation under a laser confocal microscopy, it was found that the wild strain formed a thick layer of mature biofilm at 16 h. Subsequently, the formed biofilm began to dissociate until 120 h; In contrast, a thin biofilm was formed by icaA/D genes deletion strain at 16 h after culture, and completely dissociated at 48 h. Compared with the wild strain, the MICs of 10 tested antimicrobial agents to icaA/D genes deletion strain were decreased, and the drug sensitivity of icaA/D genes deletion strain to 8 tested antimicrobial agents changed from resistance or intermediation to intermediation or sensitivity. [Conclusion] icaA/D gene deletion can significantly reduce the biofilm-formation ability and drug-resistance of MRSA.