Background and Objectives Bone marrow mesenchymal stem cells (BMSCs) show considerable promise in regenerative medicine. Many studies demonstrated that BMSCs cultured in vitro were highly heterogeneous and composed of diverse cell subpopulations, which may be the basis of their multiple biological characteristics. However, the exact cell subpopulations that make up BMSCs are still unknown. Methods and Results In this study, we used single-cell RNA sequencing (scRNA-Seq) to divide 6,514 BMSCs into three clusters. The number and corresponding proportion of cells in clusters 1 to 3 were 3,766 (57.81%), 1,720 (26.40%), and 1,028 (15.78%). The gene expression profile and function of the cells in the same cluster were similar. The vast majority of cells expressed the markers defining BMSCs by flow cytometry and gene expression analysis. Each cluster had at least 20 differentially expressed genes (DEGs). We conducted Gene Ontology enrichment analysis on the top 20 DEGs of each cluster and found that the three clusters had different functions, which were related to self-renewal, multilineage differentiation and cytokine secretion, respectively. In addition, the function of the top 20 DEGs of each cluster was checked by the National Center for Biotechnology Information gene database to further verify our hypothesis. Conclusions This study indicated that scRNA-Seq can be used to divide BMSCs into different subpopulations, demonstrating the heterogeneity of BMSCs.