A role of rhamnolipid biosurfactant to enhance the biodegradation of hydrocarbons is known to be enhancing bacterial cell surface hydrophobicity (CSH) and adhesion of cells to hydrocarbons. Assumptions regarding the mechanism for rhamnolipid in changing CSH of Gram-negative bacteria are rhamnolipid-induced release of lipopolysaccharide (LPS) from the cell's outer membrane and adsorption/orientation of rhamnolipid on the cell surface. In this study, the relation between cell-wall LPS or rhamnolipid content and CSH of a Pseudomonas aeruginosa bacterium subjected to rhamnolipid treatment was investigated to add insights to the mechanism. Results showed that the initial CSH was determined by the type of substrate the cells grow on and the stage of growth. For glucose-grown cells with low initial CSH and high LPS content, rhamnolipid sorption in cell wall had no discernable effect on CSH. For cells grown on glycerol with medium initial CSH and low LPS content, rhamnolipid sorption increased CSH of exponential-phase cells but decreased that of stationary-phase cells. For hexadecane-grown cells with high initial CSH and high LPS content, rhamnolipid sorption decreased CSH of both exponential-phase and stationary-phase cells. The results indicated that CSH has a better correlation to the content of rhamnolipid in the cell wall than to the content of LPS in the presence of rhamnolipid treatment and that rhamnolipid adsorption may be an important mechanism for rhamnolipid to alter CSH of P. aeruginosa.