Aims: Our goal was to identify a novel biosurfactant produced by a marine oil-degrading bacterium. Methods and Results: Biosurfactants were produced by Alcanivorax dieselolei strain B-5T growing with diesel oil as the sole carbon and energy source. Culture supernatant was first extracted with chloroform/methanol (1 : 1, v/v), then further purified step by step with a normal phase silica gel column, a Sephadex LH20 gel column and a preparative thin layer plate. The main component was determined to be a lipopeptide; it was chemically characterized with nuclear magnetic resonance, liquid chromatography-quadrupole ion-trap mass spectrometry, amino acid analysis and GC-MS and was found to be a mixture of proline lipids. The monomers of the proline lipids were composed of a proline residue and a fatty acid (C-14:0, C-16:0 or C-18:0). The critical micelle concentration of the mixed proline lipids was determined to be 40 mg l-1. Moreover, activity variations in ranges of pH, temperature and salinity were also detected and showed reasonable stability. Conclusions: Alcanivorax dieselolei B-5 produced a novel linear lipoamino biosurfactant, characterized as a proline lipid. Significance and Impact of the Study: A proline lipid was characterized for the first time as a bacterial biosurfactant. This product has potential in both environmental and industrial applications.