Abstract: Three cyclotides were isolated from the whole plant of Viola yedoensis in this study.The two, varv peptide E and cycloviolacin Y5, were previously reported, and a novel cycloviolacin VY1 was characterized according to the interpretation of MS/MS fragmentation of peptides which were produced from the reduced and alkylated parent peptide with the digestion of Endo Lys-C, trypsin and chymotrypsin, separately.The stability of remarkable resistance to proteolytic degradation by trypsin and chymotrypsin, and that of thermal denaturation was confirmed again.Besides, the IC₅₀ value of cycloviolacin VY1 against influenza A H1N1 virus was (2.27 ± 0.20) μg·mL^-1.It is the first cyclotide reported with anti-influenza A H1N1 virus activity in vitro assay.