Pseudomonas aeruginosa(P. aeruginosa) was a second most common commensal bacterium in healthy conjunctival sacs. When the corneal epithelial barrier is damaged, P. aeruginosa could cause keratitis, which progresses rapidly and results in corneal perforation and the loss of vision. However, the similarities and differences in characteristics between P. aeruginosa isolates from the eyes with keratitis and those from healthy conjunctival sacs are still poorly understood. In this study, four P. aeruginosa isolates from eyes with keratitis and three P. aeruginosa isolates from healthy conjunctival sacs were obtained, and genotypically characterized using Illumina high-throughput RNA sequencing. A total of 557 differentially expressed genes (DEGs) were found and included 332 upregulated genes and 225 downregulated genes in the keratitis group versus the healthy conjunctival sacs group. Of 557 DEGs, 11 DEGs analyzed with GO enrichment and the KEGG pathway were involved with the bacterial secretion system and pyoverdine metabolism and validated using quantitative reverse-transcription polymerase chain reaction. P. aeruginosa from eyes with keratitis induced more severe corneal infection and higher clinical scores in the mice. These results will contribute to develop alternative therapeutic interventions targeting virulence factors in these DEGs and facilitate the selection of therapeutic strategies.