Methamphetamine use has become a serious global public health problem and puts increasing burdens on healthcare services. Abdominal complications caused by methamphetamine use are uncommon and often go ignored by clinicians. The exact intestinal pathological alterations and transcriptomic responses associated with methamphetamine use are not well understood. This study sought to investigate the transcriptome in a methamphetamine-induced mouse model of inflammatory bowel disease (IBD) using next-generation RNA sequencing. Tissues from the ileum of methamphetamine-treated mice (n=5) and control mice (n=5) were dissected, processed and applied to RNA-sequencing. Bioinformatics and histopathological analysis were then performed. The expression profiles of intestinal tissue samples were analyzed and their expression profiles were integrated to obtain the differentially expressed genes and analyzed using bioinformatics. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the differentially expressed genes were performed using Metascape. A total of 326 differentially expressed genes were identified; of these genes, 120 were upregulated and 206 were downregulated. The Gene Ontology analysis showed that the biological processes of the differentially expressed genes were focused primarily on the regulation of cellular catabolic processes, endocytosis, and autophagy. The main cellular components included the endoplasmic and endocytic vesicles, cytoskeleton, adherens junctions, focal adhesions, cell body, and lysosomes. Molecular functions included protein transferase, GTPase and proteinase activities, actin-binding, and protein-lipid complex binding. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the differentially expressed genes were mainly involved in bacterial invasion of epithelial cells, protein processing in the endoplasmic reticulum, regulation of the actin cytoskeleton, and T-cell receptor signaling pathways. A set of overlapping genes between IBD and methamphetamine-treated intestinal tissues was discovered. The present study is the first to analyze intestinal samples from methamphetamine-treated mice using high-throughput RNA sequencing. This study revealed key molecules that might be involved in the pathogenesis of a special type of methamphetamine-induced IBD. These results offer new insights into the relationship between methamphetamine abuse and IBD. 2020 Annals of Translational Medicine. All rights reserved.