AbstractStiffness is an important physical property of biomaterials that determines stem cell fate. Guiding stem cell differentiation via stiffness modulation has been considered in tissue engineering. However, the mechanism by which material stiffness regulates stem cell differentiation into the tendon lineage remains controversial. Increasing evidence demonstrates that immune cells interact with implanted biomaterials and regulate stem cell behaviors via paracrine signaling; however, the role of this mechanism in tendon differentiation is not clear. In this study, polydimethylsiloxane (PDMS) substrates with different stiffnesses are developed, and the tenogenic differentiation of mesenchymal stem cells (MSCs) exposed to different stiffnesses and macrophage paracrine signals is investigated. The results reveal that lower stiffnesses facilitates tenogenic differentiation of MSCs, while macrophage paracrine signals at these stiffnesses suppress the differentiation. When exposed to these two stimuli, MSCs still exhibit enhanced tendon differentiation, which is further elucidated by global proteomic analysis. Following subcutaneous implantation in rats for 2 weeks, soft biomaterial induces only low inflammation and promotes tendon‐like tissue formation. In conclusion, the study demonstrates that soft, rather than stiff, material has a greater potential to guide tenogenic differentiation of stem cells, which provides comprehensive evidence for optimized bioactive scaffold design in tendon tissue engineering.