Purpose <p>The balloon vine plant, <i>Cardiospermum halicacabum,</i> has long been used as a traditional medicine for the treatment of various diseases. In the present study, the antidiabetic and antioxidant properties of a peptide identified from <i>Cardiospermum halicacabum</i> seed protein extract were investigated.</p> Methods <p><i>Cardiospermum halicacabum</i> seed protein extract was precipitated using ammonium sulfate, and an active fraction with the highest α-glucosidase inhibitory activity was purified with gel filtration column chromatography. The purified protein fraction was analyzed using SDS-PAGE, and the band obtained was subsequently analyzed using nano-liquid chromatography tandem mass spectrometry. The bioactivity score of the identified peptide was predicted using BIOPEP-UHM and PepRanker, after which it was synthesized.</p> Results <p>A 14-amino acid peptide was identified from the purified seed protein extract. The predicted bioactivity score of the peptide was more than 0.50, indicating potential bioactivity. The synthesized peptide exhibited high α-glucosidase inhibitory activity. In vitro experiments demonstrated the ability of the synthesized peptide to enhance insulin secretion and improve antioxidant activity. The peptide upregulated the expression of the transcription factors PPARγ and PDX1, resulting in increased expression of the insulin gene and subsequent insulin production by β-cells.</p> Conclusion <p>This study, for the first time, identified a novel bioactive peptide with antihyperglycemic and antioxidant properties from <i>Cardiospermum halicacabum</i> seed protein extract.</p>