Glucagon, a 29-amino acid peptide hormone antagonizing the action of insulin for glucose homeostasis, is clinically used for treatment of acute hypoglycemia in diabetic patients. Unfortunately, pharmaceutical formulation of glucagon requires an extremely acidic condition (pH 2.0) to ensure both chemical stability without degradation and pharmaceutical concentration of ~1 mg·ml-1. However, glucagon also tends to readily form amyloid fibrils at the acidic pH. In this study, we developed an E. coli-based recombinant production system of glucagon, as it could facilitate site-directed mutation and isotope labeling for NMR. Subsequently, complete backbone NMR assignments was achieved at different pHs, using the isotope [13C/15N]-enriched glucagon. Chemical shift analysis using CSI and TALOS implied alterations of secondary structure propensity and internal dynamics at pH 2.0, although experimental validation remains to be performed in detail. In addition, the terminal residues, S2 and T29, showed the most unusual chemical shift perturbations depending to the pH variation. Finally, it was observed that the glucagon fibrillation kinetics, which was monitored by thioflavin-T assay, changed upon S2A and T29A mutations. Therefore, it is suggested that the intermolecular interaction mediated by both termini might be associated with the glucagon fibrillation at acidic pH.