Mesenchymal stem cells (MSCs) are capable of exerting strong therapeutic potential for the treatment of supraspinatus tendon tear. However, MSC therapy remains underutilized and perhaps underrated due to the limited evidence of dynamic visualization of cellular behavior in vivo. Here, second near‐infrared fluorescence imaging with biocompatible PbS quantum dots (QDs) provides a cellular migration map and information on the biodistribution and clearance processes of three densities of intra‐articularly injected, labeled MSCs to treat supraspinatus tendon tear in mice. Intra‐articular injection avoids entrapment of MSCs by filter organs and reduces the QD‐induced organ toxicity. Notably, the MSCs share a similar migration direction, but the moderate density group is somewhat more efficient, showing the longest residence time and highest cell retention rate around the footprint during the repair stage. Furthermore, quantitative kinetic investigation demonstrates that labeled MSCs are cleared by feces and urine. Histomorphometric analysis demonstrates that the moderate density group achieves maximum therapeutic effect and labeled MSCs do not induce any injury or inflammation to major organs, which suggests that administration of too many or few MSCs may decrease their effectiveness. Such an imaging approach provides spatiotemporal evidence for response to MSC therapy in vivo, facilitating the optimization of MSC therapy.