The molecular mechanism underlying the development of vancomycin-intermediate Staphylococcus aureus (VISA) remains unclear. The abuses of antibacterial compounds lead to a change in the bacterial susceptibility patterns. Therefore, we examined the effect of Chlorhexidine (CHX) on in vitro development of VISA and reported CHX-selected VISA mutant Tm1 with phenotypic features similar to the clinical VISA isolates. WalKR, VraTSR, and GraSR are the most common regulatory systems involved in VISA evaluation. The expression of these systems, as well as walKR-regulated autolysins and VraTSR-regulated cell wall stimulon, were compared, by RT-qPCR, between the mutant and parental strains. The results revealed the downregulation of walKR, vraTSR, atlA, sle1, lytM, and pbpB genes in Tm1. The complete sequences of walKR and vraTSR genes was compared using the Sanger sequencing method. We detected Walk.R55C, WalR.A38T, and VraS·N340-D347del novel mutations in Tm1. These mutations were classified as deleterious mutations and predicted to affect protein function using the SIFT prediction algorithm. Novel mutations in Tm1 confirm the genetic diversity of VISA isolates. We suggest that WalKR and VraTSR may be involved in sense and response to CHX. In this regard, CHX may have a role in cell wall degradation of S. aureus and the emergence of VISA due to mutations in the CA domain of the Walk and VraS and the REC domain of WalR. Therefore, CHX should be used with caution.