Background <p>Extrachromosomal circular DNA (eccDNA) plays a significant role in cancer development. However, our understanding of its role in normal cells is limited. This study aimed to explore the roles of eccDNA in the differentiation of human bone marrow mesenchymal stem cells (BMSCs).</p> Methods <p>Through circular DNA sequencing (Circle-seq) and computational analysis, we documented eccDNAs in human BMSCs and their differentiation into osteoblasts, adipocytes, and chondrocytes. Next, the assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) data were integrated with Circle-seq data. The roles of phosphatidylinositol 4-kinase alpha (PI4KA) and eccDNA as functional enhancers in human BMSC differentiation were assessed in vitro.</p> Results <p>Our results demonstrate that eccDNA is common in human BMSCs. In the differentiated groups including osteoblasts, adipocytes and chondrocytes, eccDNA-encoded genes presented higher expression compared to the background. We found eccDNA displayed enhanced chromatin accessibility in human BMSCs, which contribute to increased transcription of genes, such as runt-related transcription factor 2 (RUNX2), a key transcription factor for BMSC osteogenesis. We further found PI4KA, an eccDNA-encoded gene, promoted osteogenic differentiation of human BMSCs via the PI3K/AKT/mTOR pathway. Finally, we demonstrated eccDNA as functional enhancers to regulate BMSC differentiation in a dose-dependent manner.</p> Conclusions <p>Our study provides the evidence of eccDNA regulating BMSC differentiation and highlights the roles of eccDNA as transcription template and enhancer in normal cells, which will facilitate future research and clinical applications.</p>