Amide bonds in peptide macrocycles have inherent polar properties which limit their application in medicine. Desolvation of the amide bond by masking of amide hydrogen bond donor (HBD) has been reported to enhance membrane permeability. We demonstrate that shielding the amide hydrogen bond acceptor (HBA) by substituting an oxygen atom of the backbone amide with sulfur (>C=O to >C=S) results in enhanced lipophilicity, conformational rigidity & permeability using model compounds with varying ring sizes and properties. We also show that peptide macrocycles are susceptible to proteolysis by enzymes in the gastrointestinal fluids, against which thioamide substitution confers significant protection, which improves the bioavailability of a macrocyclic peptide composed of hydrophobic amino acid residues when administered through the oral route in a rat model. To show the robustness of our modification we compared the permeability, stability & oral bioavailability of thioamidated analogs with the most dominant backbone modification, N-methylation. Furthermore, thioamidation of a bioactive macrocyclic peptide composed of polar amino acid residues resulted in analogs with prolonged plasma exposure due to its enhanced plasma protein binding and plasma stability. As an outcome, we observe a longer duration of drug 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.