[Objective] This study aimed to investigate the effect of modified mouse host defense peptide CRAMP on the mature biofilm of Pseudomonas aeruginosa PAO1 srtain based on RNA-Seq technology.[Methods] Crystal violet method was used to detect the biofilm biomass, and laser confocal scanning microscope (CLSM) was used to observe the morphological changes of biofilms. The gene expression differences at transcription level between biofilms treated with CRAMP and the non-peptide control were analyzed by the PE150 sequencing strategy using Illumina second-generation high-throughput sequencing platform. The content of alginate in PAO1 biofilms was determined by the 1,3-Dihydroxynaphthalene method.[Results] The biofilm biomass was significantly reduced by modified peptide CRAMP at the range of 0.98-62.50 μg/mL. CLSM showed that CRAMP could significantly reduce the total fluorescence intensity of biofilms. The results showed that 12636700 clean-read pairs were obtained by transcriptome sequencing. A total of 1582 differential genes were identified, including 800 genes that were up-regulated, and 782 genes were down-regulated. The GO functional enrichment analysis showed that 1226 genes were compared to the GO functional analysis database. Among these differentially expressed genes, they were related to molecular function, biological process, and cell composition. Enrichment analysis of the KEGG pathway showed that 603 differentially expressed genes were compared with 96 metabolic pathways in KEGG. There are various kinds of amino acid metabolism pathways, fatty acid metabolism pathway, tricarboxylic acid cycle, biofilm regulatory system, and so on. It was found that modified peptide CRAMP may act on PAO1 c-di-GMP system, enhance bacterial motility and biofilm dispersion, and be related to the quorum sensing (QS) system and alginate synthesis. Finally, it was verified that the modified peptide CRAMP significantly reduced the content of alginate in PAO1 biofilms.[Conclusion] The modified peptide CRAMP has an impair effect on PAO1 mature biofilms and can lead to the decrease of alginate content in mature biofilm. Transcriptomics analysis implied that CRAMP might exert its effectiveness by reducing the level of PAO1 c-di-GMP, and the mechanism remains to be further explored.