Surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a ${eta}-hydroxy$ fatty acid produced by several strains of Bacillus sp. Surfactin isoforms produced by endophytic Bacillus sp. CY22 from a balloon flower were isolated and characterized. It was found that the purified surfactin had three isoforms with protonated masses of m/z 1,008, 1,022, and 1,036, and different structures in combination with Na, K, Ca ions using MALDI-TOF MS, ESI-MS/MS, and ICP MS, respectively. In the MS/MS analysis, the isolated surfactin had the identical amino acid sequence (LLVDLL) and hydroxy fatty acids (with 13 to 15 carbons in length), even though isolated from different Bacillus strains. The sfp22 gene, required for producing the surfactin, consisted of an open reading frame (ORF) of 675 bp encoding 224 amino acid residues with a signal peptide of 20 amino acids. The predicted amino acid sequence of sfp22 was very similar to that of Ipa-8.