Aims Relatively, few anti‐biofilm polysaccharides against Pseudomonas aeruginosa were done to investigate the underlying molecular mechanism. Exopolysaccharide EPS273 can clearly reduce biofilm formation and infection of P. aeruginosa. This study aims to investigate its anti‐biofilm and anti‐infection mechanism on transcriptional level. Methods and Results Herein, we used an RNA‐Seq transcriptomic approach to investigate the underlying anti‐biofilm and anti‐infection mechanism of EPS273. The expression levels of a large number of genes were changed after P. aeruginosa PAO1 was treated with EPS273. Especially, the genes related to biofilm formation, such as gene involved in production of extracellular matrix and virulence factor, genes involved in flagella and cell motility and genes involved in iron acquisition. Notably, the expression levels of genes involved in regulatory and signal transduction were markedly downregulated, such as two‐component system PhoP‐PhoQ and quorum sensing (QS) system LasI/LasR and RhlI/RhlR. Furthermore, when genes phoP and phoQ were disrupted, respectively, the reduction of biofilm formation and cell motility in mutant △phoP or △phoQ was also detected. Conclusion EPS273 may exert its anti‐biofilm and anti‐infection function by downregulating gene expression of two‐component system PhoP‐PhoQ and QS systems LasI/LasR and RhlI/RhlR of P. aeruginosa, which further regulated expression of genes involved in biofilm formation. Significance and Impact of the Study Our data will expand understanding of anti‐biofilm mechanisms of polysaccharides on transcriptomic level.