Freeze-drying is a critical process for ensuring the long-term stability of therapeutic polypeptides. This study used all-atom molecular dynamics simulations to investigate the interactions between glucagon and β-cyclodextrin (β-CD) during a multistage process designed to mimic freeze-drying and reconstitution. Glucagon is a highly flexible 29-amino-acid peptide hormone with an inherently unstable secondary structure, and the molecular-level basis of excipient-mediated protection remains unclear. Simulations were performed using the OPLS-2005 force field and the SPC water model. The protocol included room-temperature equilibration at 300 K, cooling to 233 K, four consecutive drying stages represented by the progressive removal of water molecules, and subsequent reconstitution. Structural changes were evaluated using backbone root-mean-square deviation, radius of gyration, residue-level flexibility, dihedral-angle distributions and peptide–excipient interactions. Through specific inclusion complexation, β-CD achieved superior preservation of the local C-terminal α-helix and a functionally relevant conformation associated with glucagon receptor binding, although protection came at the expense of overall conformational stability. At the end of the fourth drying stage, the glucagon C-terminus was located inside the hydrophobic cavity of β-CD. Five hydrogen bonds were also observed between glucagon and β-CD. Local stabilisation within β-CD did not extend to the peptide as a whole. Following reconstitution, the backbone RMSD increased to approximately 6–7 Å, showing that β-CD helped preserve the C-terminal helix but did not maintain the initial global conformation. These findings provide an atomistic explanation of how β-CD may selectively protect structural regions of glucagon through supramolecular host-guest interactions during freeze-drying-related stresses.