Glucagon-like peptide-1 (GLP-1) is an attractive lead for antidiabetic drug development but its utility is limited due to poor in vivo stability. In this study, we developed a C-terminal site-specific PEGylated analog of GLP-1 based on the structure-activity relationship of GLP-1. Using three steps of chromatography, we obtained the conjugate with a high purity. Polyethylene glycol (PEG) conjugation greatly enhanced GLP-1 potency in vivo without changing its original conformation. In streptozotosin diabetic mice, this conjugate had an improved antidiabetic effect, as measured by fasting glucose/insulin/glycosylated serum protein, oral glucose tolerance test, and histochemical studies. Additionally, this PEG-conjugated peptide delayed gastric emptying without significant effects on body weight gain. Our data indicate that this conjugate may be a promising lead for the development of antidiabetic drugs.