Antagonism of glucagon's biological action is a proven strategy for decreasing glucose in diabetic animals and patients. To achieve full, potent, and selective suppression, we chemically optimized N-terminally truncated glucagon fragments for the identification and establishment of the minimum sequence peptide, [Glu9]glucagon(6-29) amide (11) as a full antagonist in cellular signaling and receptor binding (IC50 = 36 nM). Substitution of Phe6 with l-3-phenyllactic acid (Pla) produced [Pla6, Glu9]glucagon(6-29) amide (21), resulting in a 3-fold improvement in receptor binding (IC50 = 12 nM) and enhanced antagonist potency. Further substitution of Glu9 and Asn28 with aspartic acid yielded [Pla6, Asp28]glucagon amide (26), which demonstrated a further increase in inhibitory potency (IC50 = 9 nM), and improved aqueous solubility. Peptide 26 and a palmitoylated analogue, [Pla6, Lys10(γGluγGlu-C16), Asp28]glucagon(6-29) amide (31), displayed sustained duration in vivo action that successfully reversed glucagon-induced glucose elevation in mice.