Osteoblasts actively generate cell traction force (CTF) to sense chemical and mechanical microenvironments. Fluid shear stress (FSS) is a principle mechanical stimulus for bone modeling/remodeling. FSS and CTF share common interconnected elements for force transmission, among which the role of the protein-material interfacial force (F-ad) remains unclear. Here, we found that, on the low F-ad surface (5.47 +/- 1.31 pN/FN), CTF overwhelmed F-ad to partially desorb FN, and FSS exacerbated the desorption, resulting in disassembly of the actin cytoskeleton and focal adhesions (FM) to reduce CTF and establishment of a new mechanical balance at the FN-material interface. Contrarily, on the high F-ad surface (27.68 +/- 5.24 pN/FN), pure CTF or the combination of CTF and FSS induced no FN desorption, and FSS promoted assembly of actin cytoskeletons and disassembly of FAs, regaining new mechanical balance at the cell-FN interface. These results indicate that F-ad is a mechanical regulator for transmission of CTF and FSS, which has never been reported before.