The interfacial behavior of surfactin, a microbial lipopeptide, was investigated. The surface pressure-molecular area (π-A) isotherms were recorded as a function of compression speed. It was found that the π-A isotherms on subphase at pH＝2 were compression-speed dependent, indicating that the isotherms are under non-equilibrium in the condition. The LB films of surfactin monolayer under different compression speeds were imaged by atomic force microscopy (AFM). The AFM images showed that surfactin molecules could form homogeneous quasi-circular aggregates under the moderate compression speed (0.6 nm28226;mol－18226;min－1), while they gathered into surface clusters under other investigated speeds. Based on the results presented here, a mechanism by which molecules in monolayer would self-assemble into surface aggregates was proposed.