Skeletal transverse harmony between maxilla and mandible is essential for balanced vertical and sagittal dental and skeletal relationship, as well as facial esthetics and periodontal health. However, patients, especially adult ones, responded to maxillary expansion differently. It remains unclear why some cases fail while others succeed despite usage of miniscrews for anchorage reinforcement. A deep understanding of the biological basis underlying maxillary expansion is the fundament to answer this question. Unfortunately, the scientific evidence behind the maxillary expansion is largely missing. Cell population within mid-palatal suture and cellular and molecular mechanisms in response to mechanical expansion forces are potential factors other than the differences in calcification patterns of the mid-palatal suture, craniofacial architecture, and patient’s age. Mesenchymal stem cell (MSC) refers to a group of heterogeneous multipotent stem cells that can self-renew and further differentiate into several cell types, including osteoblasts, chondrocytes, and adipocytes, which plays an important role in bone remodeling. We show here that suture-MSCs were labeled by red fluoresces by crossing Gli1-CreERT2 mice with Cre reporter tdTomato mice. Maxillary expansion seems to promote the proliferation of the Gli1+ suture-MSCs. Single cell transcriptomics identified MSC lineage trajectories shift in response to maxillary expansion. Interestingly, we found the immune cells, like neutrophils, also under active immunomodulation. In summary, our data proved the presence of MSCs within mid-palatal suture and MSC lineage trajectories and their unique immune microenvironment have been revealed by single cell transcriptomics. This work may provide a more precise immune-MSC modulatory target for maxillary expansion.