Vancomycin-intermediate Staphylococcus aureus (VISA) is generated from vancomycin-susceptible Staphylococcus aureus by multiple spontaneous mutations. We previously reported that sequential acquisition of mutations in the two-component regulatory systems vraSR and graRS was responsible for the VISA phenotype of strain Mu50. Here we report on the identification of a novel set of regulator mutations, a deletion mutation in two-component regulatory system walRK (synonyms, vicRK and yycFG), and a truncating mutation in a proteolytic regulatory gene, clpP, responsible for the raised vancomycin resistance in a laboratory-derived VISA strain, LR5P1-V3. The contributory effect of the two mutations to vancomycin resistance was confirmed by introducing the walK and clpP mutations into the vancomycin-susceptible parent strain N315LR5P1 by a gene replacement procedure. The vancomycin MIC of N315LR5P1 was raised from 1 to 2 mg/liter by the introduction of the walK or clpP mutation, but it was raised to 4 mg/liter by the introduction of both the walK and clpP mutations. The vancomycin MIC value of the double mutant was equivalent to that of strain LR5P1-V3. Like VISA clinical strains, LR5P1-V3 and the double mutant strain LR5P1walK*clpP* exhibited a thickened cell wall, slow growth, and decreased autolytic activity. Transcriptional profiles of the mutants with gene replacements demonstrated that introduction of both the walK and clpP mutations could alter expression of dozens or hundreds of genes, including those involved in cell envelope and cellular processes, intermediary metabolism, and information pathway. A mutation prevalence study performed on 39 worldwide clinical VISA strains showed that 61.5, 7.7, 10.3, and 20.5% of VISA strains harbored mutations in walRK, clpP, graRS, and vraSR, respectively. The mutation of walRK was most frequently carried by VISA strains. Together, these results suggested that the mutations of walK and clpP identified in LR5P1-V3 constitute a new combination of genetic events causing vancomycin resistance in Staphylococcus aureus.