[Objective] Lipopeptides (LPs) are important biosurfactants produced by microorganisms and have significant influences on bacterial biological functions and broad-spectrum antagonistic abilities towards many botanic and human pathogens. LPs produced by Pseudomonas chlororaphis are not reported as yet. [Methods] This study predicted the composition and sequence of the amino acids within the LP produced by P. chlororaphis HT66. The LP biosynthetic genes deletion strain HT66Δclp was constructed, and subsequently the LP produced by strain HT66 was analyzed by the UPLC/QTOF-MS according to the lost metabolite in the mutant. At last, the roles of the LP on bacterial growth, phenazine-1-carboxamide (PCN) production, biofilm formation and swarming motility were studied. [Results] The amino acid sequence of the LP produced by strain HT66 was predicted to be L-Leu–D-Glu–D-allo-Thr–D-Val–L-Leu–D-Ser–L-Leu–D-Ser–L-Ile and the LP was verified to be viscosin via comparing the mass spectra data of the metabolites in the wild type and the mutant. Deficiency of LP was found to have no distinct effect on bacterial growth of strain HT66, however, the reduction in PCN production, biofilm formation and swarming motility were observed in the mutant. [Conclusion] The LP produced by strain HT66 is viscosin and it regulates several biological functions including metabolism, biofilm formation and motility. This study reported the structure and function of an LP produced by P. chlororaphis, which may be helpful in learning its synthesis and regulation mechanism and lay a foundation for its development and application.