Microsatellite instability (MSI) has emerged as one of the key biological features of colorectal cancer (CRC). However, controversies remain regarding the association between the MSI status and clinicopathological characteristics of CRC. Therefore, it is crucial to identify potential key genes and pathways associated with MSI in CRC. In the present study, the GSE25071 gene expression profile was retrieved, with thirty‑eight cases of microsatellite stable (MSS), five of MSI‑High (MSI‑H) and three of MSI‑Low (MSI‑L) CRC patients. The differentially expressed genes (DEGs) were analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes pathway enrichment, gene set enrichment analysis (GSEA) and gene co‑expression network analysis. Weighted gene correlation network analysis (WGCNA) was used for the gene modules and correlation of clinical traits. A total of forty‑nine DEGs were identified between MSI‑H and MSS, including six upregulated and forty‑three downregulated DEGs. Only the DEGs of MSI‑H and MSS were subjected to subsequent analysis (limited number of DEGs of MSI‑L and MSS, MSI‑H and MSI‑L). RNA metabolic process, endoplasmic reticulum and chemokine receptor binding were the top ranked terms in GO enrichment. The hub genes of co‑expression network of DEGs included zinc finger protein (ZNF) 813, ZNF426, ZNF611, ZNF320 and ZNF573. The GSEA of MSI‑H and MSS indicated that the mammalian target of rapamycin complex 1 signaling was significantly enriched with a nominal P‑value of 0.038 and normalized enrichment score of 0.446. The WGCNA results showed that the pink module was the top in correlation with MSI status (R2=0.5, P=0.0004). The genes in the pink module were significantly enriched in proteins targeting to endoplasmic reticulum, cytosolic part, structural constituent of ribosome and ribosome pathway. The hub genes identified in the pink module were ribosomal protein L12 (RPL12), RPS3A, RPS9, RPL27A, RPL7, RPL28, RPL14, RPS17, mitochondrial ribosomal protein L16, and G elongation factor, mitochondrial 2. The present study identified key genes and pathways associated with MSI, providing insightful mechanisms.