The emergence of bacteria resistant to vancomycin, often the antibiotic of last resort, poses a major health problem. Vancomycin-resistant bacteria sense a glycopeptide antibiotic challenge and remodel their cell wall precursor peptidoglycan terminus from d-Ala-d-Ala to d-Ala-d-Lac, reducing the binding of vancomycin to its target 1000-fold and accounting for the loss in antimicrobial activity. Here, we report [Ψ[C(═NH)NH]Tpg(4)]vancomycin aglycon designed to exhibit the dual binding to d-Ala-d-Ala and d-Ala-d-Lac needed to reinstate activity against vancomycin-resistant bacteria. Its binding to a model d-Ala-d-Ala ligand was found to be only 2-fold less than vancomycin aglycon and this affinity was maintained with a model d-Ala-d-Lac ligand, representing a 600-fold increase relative to vancomycin aglycon. Accurately reflecting these binding characteristics, it exhibits potent antimicrobial activity against vancomycin-resistant bacteria (MIC = 0.31 μg/mL, VanA VRE). Thus, a complementary single atom exchange in the vancomycin core structure (O → NH) to counter the single atom exchange in the cell wall precursors of resistant bacteria (NH → O) reinstates potent antimicrobial activity and charts a rational path forward for the development of antibiotics for the treatment of vancomycin-resistant bacterial infections.