Objective To explore gene expression profiling of colon tissues from mesalazine-treated inflammatory bowel disease (IBD) mice using RNA-sequencing (RNA-seq), and provide a better understanding of the mechanism of mesalazine (5-ASA) in the treatment of IBD. Methods Dextran sodium sulfate (DSS) was used to establish the IBD mouse model. Fifteen mice (C57BL/ 6) were randomly assigned to three groups: control (n= 5), DSS (n= 5, using DSS to induce IBD), and DSS+5-ASA (n= 5, using 5-ASA to treat IBD). Three mice per group were randomly selected and colon tissues extracted for RNA-seq. Bioinformatic analysis was used for two comparisons (DSS vs. control, and DSS+5- ASA vs. DSS), and differentially expressed genes (DEGs) and biological signal pathways were identified. The top DEGs were further examined using microarray data of biopsies from ulcerative colitis patients and those treated with 5-ASA. Results Bioinformatic analysis identified 2983 DEGs, including 604 up-regulated and 753 down-regulated genes in the DSS+5-ASA group and DSS group comparison. In the DSS group and control group comparison, there were 1626 DEGs, 820 of which were up-regulated and 806 were down-regulated. Gene ontology and KEGG analysis of these DEGs suggested certain biological processes and pathways. The expression patterns of top DEG candidates, such as Rnase1 and Cxcl10, were confirmed in patients’ samples. Conclusions By exploring gene expression profiles in colon tissues from 5-ASA- treated IBD mice using RNA-seq and human mucosal biopsies, this study identified genes and pathways involved in the treatment process of IBD, providing new insights for understanding the mechanism of 5-ASA in IBD treatment.