Background <p>Mesenchymal stem cells (MSCs) within the shoulder joint serve as a critical progenitor pool for regenerating the fibrocartilaginous enthesis during rotator cuff repair. However, the chondrogenic potential of MSCs across distinct rotator cuff sites, along with the underlying molecular mechanisms, remains to be elucidated.</p> Methods <p>We performed a comparative analysis of human subacromial bursa-derived MSCs (sMSCs) and rotator cuff enthesis-derived MSCs (rMSCs). By integrating functional chondrogenic assays with longitudinal bulk and single-cell RNA sequencing, we reconstructed source-specific differentiation trajectories and identified key regulatory drivers.</p> Results <p>rMSCs exhibited superior chondrogenic potency compared to sMSCs. Transcriptional profiling identified a region-specific pro-chondrogenic module in rMSCs driven by the transcription factor SIX2. Single-cell atlas construction revealed that tissue origin dictates lineage fate: rMSCs were enriched for a high-potency SIX2⁺ progenitor population that differentiated into metabolic-active S100A2⁺ chondrocytes. Conversely, sMSCs were dominated by DPP4⁺ progenitors that preferentially bifurcated into an aberrant CXCL8⁺ inflammatory trajectory. Mechanistically, we demonstrated that SIX2 acted as a competency factor, coordinating early proliferative expansion with late-stage matrix assembly and, crucially, active suppression of inflammatory signaling. Accordingly, lentiviral overexpression of SIX2 in sMSCs was sufficient to rescue their chondrogenic defects and rewire their trajectory toward a regenerative phenotype.</p> Conclusions <p>Our findings define the transcriptional hierarchy of shoulder-resident progenitors, identifying SIX2 as a master regulator that couples chondrogenesis with immune evasion. This establishes a molecular framework for precision cell sourcing and rational design of lineage-specific therapies.</p> Graphic abstract <p></p>