Rhamnolipid biosurfactant plays an important role in accelerated bioremediation of hydrocarbon-contaminated sites. In this article, the in vitro effects of rhamnolipid biosurfactant on reduction of energy consumption of biodesulfurization activity were evaluated in recombinant Pseudomonas aeruginosa ATCC9027. The data obtained of these analyses showed that the amount of 2-hydroxybiphenyl production in the presence of rhamnolipid was approximately 150% higher than that produced without rhamnolipid. These analyses demonstrated that the rhamnolipid is a major cause of reduction of energy consumption in biodesulfurization of resting cells. However, the rhamnolipid had no effect in reduction of energy consumption in biodesulfurization of growing cells.