Collagen is the most abundant protein in animals. Every third residue in a collagen strand is a glycine with φ, ψ = −70°, 175°. A recent computational study suggested that replacing these glycine residues with d-alanine or d-serine would stabilize the collagen triple helix. This hypothesis is of substantial importance, as the glycine residues in collagen constitute nearly 10% of the amino acid residues in humans. To test this hypothesis, we synthesized a series of collagen mimic peptides that contain one or more d-alanine or d-serine residues replacing the canonical glycine residues. Circular dichroism spectroscopy and thermal denaturation experiments indicated clearly that the substitution of glycine with d-alanine or d-serine greatly disfavors the formation of a triple helix. Host–guest studies also revealed that replacing a single glycine residue with d-alanine is more destabilizing than is its replacement with l-alanine, a substitution that results from a common mutation in patients with collagen-related diseases. These data indicate that the glycine residues in collagen are not a surrogate for a d-amino acid and support the notion that the main-chain torsion angles of a glycine residue in the native structure (especially, φ > 0°) are critical determinants for its beneficial substitution with a d-amino acid in a protein.