Solvent shielding of the amide hydrogen bond donor (NH groups) through chemical modification or conformational control has been successfully utilized to impart membrane permeability to macrocyclic peptides. We demonstrate that passive membrane permeability can also be conferred by masking the amide hydrogen bond acceptor (>C=O) through a thioamide substitution (>C=S). The membrane permeability is a consequence of the lower desolvation penalty of the macrocycle resulting from a concerted effect of conformational restriction, local desolvation of the thioamide bond, and solvent shielding of the amide NH groups. We also show that peptide macrocycles are susceptible to proteolysis by enzymes in the gastrointestinal fluids, against which thioamide substitution confers significant protection. The enhanced permeability and metabolic stability on thioamidation improved the bioavailability of a macrocyclic peptide composed of hydrophobic amino acid residues when administered through the oral route in a rat model. In comparison, thioamidation of a bioactive macrocyclic peptide composed of polar amino acid residues resulted in analogs with prolonged plasma exposure and a longer duration of action in rats when delivered through the subcutaneous route. Thus, highlighting the potential of O to S substitution as a chemically stable backbone modification in improving the pharmacological properties of peptide macrocycles.