[Objective] In order to study the mechanism of vancomycin intermediate resistance Staphylococcus aureus development, strains were treated with vancomycin in vitro and mutations in critical genes were analyzed. [Methods] Thirteen vancomycin susceptible S. aureus were treated by low dose of vancomycin in vitro. Minimum inhibitory concentrations were determined by the agar dilution and E-test methods. The complete sequences of rpoB, vraS, graR and graS associated with drug-resistance development were compared with those in the parental strains. [Results] Through a 60 d vancomycin treatment in vitro, 6 vancomycin intermediate resistance strains were generated, whereas 7 strains remained vancomycin susceptible. Sequence analysis revealed that 3 vancomycin intermediate resistance strains contained L466S and H481N mutations in rpoB gene and R232K mutation in graS gene. [Conclusion] Evolution of vancomycin intermediate resistance S. aureus could be achieved by long-term vancomycin treatment in vitro. Our results suggest that rpoB and graS play crucial roles in vancomycin non-susceptibility development, whereas vraS and graR are less important.