Objective To explore the effect of phosphodiesterase PA4781 on bacterial motility and virulence. Methods Motility of bacteria was detected by measuring the diameter of bacterial lawn, the ability of bacterial biofilm to secrete extracellular polysaccharides was detected by Congo-red staining and phenol-sulphate colorimetry. The expression levels of mRNA of twitching motility-mediating gene PilZ, extracellular polysaccharide synthesis relevant gene PelA and pyocyanin-controlling gene PhzM were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Extracellular DNA produced by bacteria during different periods was detected by agarose gel electrophoresis, the overall activity of extracellular protease was detected by measuring the diameter of transparent circle of plaque in milk plate. Results Compared with PA03 wild-type strain, motility and mRNA expression of PilZ of PA4781 knockout strain decreased, motility and mRNA expression of PilZ of PA4781 overexpressed strain increased, differences were both significant (both P < 0.05); compared with PA03 wild-type strain, secretion of extracellular polysaccharide and mRNA expression of PelA of PA4781 knockout strain increased, secretion of extracellular polysaccharide and mRNA expression of PelA of PA4781 overexpressed strain decreased, diffe-rences were both significant (both P < 0.05); extracellular DNA secretion, expression of pyocyanine gene PhzM, and hydrolysis ability of extracellular protease of PA4781 knockout strain and overexpressed strain were all lower than PA03 wild-type strain, differences were all significant (all P < 0.05). Conclusion PA4781 may promote the motility of Pseudomonas aeruginosa by promoting the expression of gene PilZ. PA4781 may inhibit the secretion of extracellular polysaccharide by inhibiting the expression of gene PelA. PA4781 may not directly affect the secretion of extracellular DNA, as well as the production of virulence factor pyocyanine and extracellular protease.