In this study, we found that tendon stiffness increased following injury in a rat tendon injury model, indicating the potential effect of stiffness on tendon regeneration. Then, we collected the conditioned medium (CM) of TSPCs cultured on matrices with different stiffnesses, and cultured TSPCs and macrophages with CM to evaluate the impact of stiffness on intercellular communications within tendon. Mass spectrometry-based proteomics and secretomes were performed to discern the underlying mechanism of the stiffness regulating intercellular crosstalks.