[Background] There is a gene PA0575 related to cyclic-di-guanosine monophosphate (c-di-GMP) metabolism in Pseudomonas aeruginosa PAO1. [Objective] To investigate the effect of c-di-GMP metabolism related gene PA0575 on phenotype of PAO1. [Methods] Identification of genetic background of strains by PCR method. Swimming, swarming, twiching and biofilm were used to analyze the phenotype of transposon mutant strain and wild type PAO1, and antibiotics were added to motility medium to study the effect of antibiotics on motility. The fusion protein expression vector of PA0575 gene was constructed and the prokaryotic expression of the protein was induced. [Results] Inconsistency of transposon insertion mutation sites among three mutant strains, and the mutation sites are inconsistent. The results of intracellular c-di-GMP level test showed that: The c-di-GMP content of PA0575-1 strain was higher than that of wild type PAO1 strain (P<0.05). In the motility test, compared with the wild type PAO1 strain, enhanced Swimming of strain PA0575-1 (P<0.05). The swimming motility and swarming motility of PA0575-2 and PA0575-3 were enhanced (P<0.05). The effect of adding four antibiotics to motility media showed that three different insertion sites led to the inhibition of chloramphenicol on exercise ability. The results of biofilm detection showed that after 18 hours of bacterial culture, the biofilm content of PA0575-1 was significantly lower than that of wild type PAO1 (P<0.05). Eight expression vectors of PA0575 gene were successfully constructed and induced. SDS-PAGE results showed that the expression vector was induced by IPTG to obtain heterologous expression protein. [Conclusion] PA0575 gene decreased the level of intracellular c-di-GMP of Pseudomonas aeruginosa, affected the phenotype and inhibited the expression of chloramphenicol resistance genes. The above studies laid the foundation for the effect of PA0575 gene on phenotype.