Background Our previous study revealed that the virulence-associated protein E (virE) gene of methicillin-resistant Staphylococcus aureus (MRSA) was highly expressed in virulent clonal strains. However, systematic reports on the MRSA virE gene have not yet been conducted. Bioinformatics analysis and cloning of the virE gene were performed. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was used to construct a virE gene knockout of the YNSA7 strain, and its effect on strain virulence was analysed. The bacterial growth, biofilm formation ability and cytotoxicity of the wild type (WT) and Delta virE strains were compared. The aim of this study was to explore the structure and function of the virE gene of MRSA, and to provide a basis for subsequent in-depth study of this gene. Results BLAST and alignment analyses revealed that the virE gene was identified in both community- and hospital-acquired MRSA strains and was a highly conserved virulence-associated protein factor of S. aureus. The protein with the highest similarity to its predicted amino acid sequence was a pathogenicity island protein of Staphylococcus saccharolyticus, with a similarity of 73.42%. The SWISS-MODEL protein structure prediction demonstrated that the VirE protein was mainly an alpha-helical structure, and protein interaction analysis showed that the VirE protein and phage-related proteins formed an interaction network. The VirE protein was successfully cloned and expressed. The bacteria-cell interaction indicated that VirE and the clonal strain could cause more extensive cytopathic and necrotic effects in Hep-2 cells. Compared with the WT strain, the Delta virE strain presented a growth delay from 6 h to 12 h. No significant difference in biofilm formation capacity was detected between the WT and Delta virE strains. Compared with that of the wild-type strain, the virulence of the knockout strain was decreased in both Hep-2 and RAW264.7 cells. Conclusion The virE gene has been characterized in multiple ways by bioinformatics, clonal expression and knockout experiments. Cytotoxicity to cells indicated that the virE gene is a potential virulence factor in MRSA.