Extracellular electron transfer is the predominant electricity generation process in microbial fuel cells (MFCs). Our pervious study have proved that the anodic adsorption of rhamnolipid resulted in the Frumkin effect, which enhanced anodic microorganism attachment and accelerated anodic electron transfer. In this study, an in-depth research on the influence of rhamnolipid on functional genes of anodic biofilms metagenomes was carried out to explain its mechanism at the gene level. The result showed that the composition and distribution of functional genes in each dominant genus were different. The category of signal transduction mechanisms was the dominant function category in exoelectrogens, and its relative abundance in the metagenome significantly increased from 4.56 to 5.86% from rhamnolipid addition. Additionally, the metabolic pathway and electron flow analysis revealed that electron flows tend to choose direct electron transfer in the presence of rhamnolipids, and resulting in the increase of Coulombic efficiency from 19.10±0.79% to 27.39±1.07%.