Abstract: Heterogeneous vancomycin-intermediate Staphylococcus aureus （hVISA） harbors minor resistant subpopulations that routinely evade detection by conventional susceptibility testing and represent a principal driver of clinical failure in vancomycin-based therapy. This organism develops intermediate resistance through a multilayered regulatory network: crosstalk between the WalKR and GraSR two-component systems hyperactivates peptidoglycan biosynthesis, leading to pronounced cell wall thickening that serves as a physical barrier to antibiotic penetration. Under antimicrobial stress, the strain initiates metabolic reprogramming of amino acid and purine pathways; in parallel, the mgrA-agr regulatory axis, acting in concert with biofilm formation, synergistically enhances both resistance and virulence. Sustained vancomycin selection drives convergent evolution, generating stably transmissible resistant clones that simultaneously exhibit cross-resistance to lipoglycopeptides and collateral sensitivity to β-lactams. The population analysis profile-area under the curve （PAP-AUC） remains the reference standard for detection; however, its labor-intensive procedure limits rapid screening applications. Emerging techniques, particularly single-cell sequencing and CRISPR-Cas9-based genome-wide screening, offer new opportunities for dissecting the mechanisms of hVISA heteroresistance. On the basis of available evidence, this review proposes two conceptual models—the "cell wall stress-threshold response" model and the "metabolic burden-collateral sensitivity trade-off" model—to explain the emergence of resistant subpopulations and the collateral sensitivity phenotype, respectively. An integrative systems-biology approach to deciphering multilayer regulatory networks is expected to provide a theoretical framework for optimizing rapid diagnostic algorithms, identifying novel anti-staphylococcal targets, and formulating evidence-based infection control strategies.