BACKGROUND: Gastrointestinal mesenchymal stromal tumors (GISTs) are a group of intramural tumors that exhibit a wide range of morphologies. Dysfunction or loss of interstitial cells of Cajal (ICCs) is correlated with the disorders of gastrointestinal motility. At present, the characterization and molecular mechanisms underlying the role of ICCs in GIST are still not clear. METHODS: The GSE162115 dataset from Gene Expression Omnibus database was processed using Seurat package for quality control, data normalization, and cell clustering. Differential expression and functional enrichment analyses were performed using the FindAllMarkers function and clusterProfiler package. Cellular heterogeneity was assessed by CytoTRACE and potential regulatory mechanisms of ICCs in GISTs were investigated using SCENIC. Cellular communication was inferred and analyzed applying the CellChat package. RESULTS: Eight clusters were identified based on 34,861 cells. Intra-tumor samples had a higher proportion of ICCs than peri-tumor. ICCs were related to cell cycle and glycolytic activity in intra-tumor samples, while those in peri-tumor samples were involved in immune response. Further analysis identified four ICC subgroups (subcluster 1-4), of which subcluster 3 showed the most typical stem cell properties and interacted with the rest of the cells through the MIF-CD74 (CD44) protein. CONCLUSION: This study analyzed the heterogeneity and stem cell properties of ICCs in GISTs, revealing the molecular mechanisms and potential therapeutic targets for GISTs.