The effect of rhamnolipids on pyrene degradation by Pseudomonas sp. GP3A was investigated to explore the interrelationships of biosurfactant, hydrophobic substrate and degrading bacteria. The cell surface hydrophobicity, lipopolysaccharide, hydrogen bond, bacterial biomass and pyrene degradation were determined. The results showed that the apparent solubility of pyrene was enhanced significantly when the concentration of rhamnolipids was higher than critical micelle concentration (CMC) of 60 mg x L(-1). Biosurfactant can increase the cell surface hydrophobicity by releasing the component of the cell wall-lipopolysaccharide; With the increase of cell surface hydrophobicity from 12% to 55%, bacterial biomass increased from 4.4 x 10(6) CFU x mL(-1) to 1.2 x 10(7) CFU x mL(-1), corresponding with the increase of pyrene degradation from 16% to 44%. Meanwhile, hydrogen bond was formed between biosurfactant and bacteria, which was beneficial to hydrophobic substrate degradation. The half-life of pyrene was shorten significantly. The average residual rate of pyrene in 10 days was 81% without rhamnolipids, but decreased to 57%, 41%, 33% and 26%, respectively with the addition of 20, 50, 200 and 500 mg x L(-1) of rhamnolipids.