Background: Proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 is crucial for normal hemostasis. Our previous studies demonstrate that binding of coagulation factor VIII (or FVIII) to VWF enhances the proteolytic cleavage of VWF by ADAMTS13 under shear. Objectives: Present study aims to determine the mechanism underlying FVIII-mediated enhancing effect on VWF proteolysis by ADAMTS13 under force. Methods: Single molecular force spectroscopy, atomic force microscopy, and surface plasmon resonance are all used. Results: Using single molecule force spectroscopy, we show that an addition of FVIII (~5 nmol/L) to D′D3 or D′D3A1 does not significantly alter force-induced unfolding of these fragments; however, an addition of FVIII at the same concentration to D′D3A1A2 eliminates its long unfolding event at ~40 nm, suggesting that binding of FVIII to D′D3 and/or A2 may result in force-induced conformational changes in A2 domain. Atomic force spectroscopy further demonstrates the direct binding between FVIII and D′D3 (or A2) with an intrinsic 2-dimensional off-rate (k0) of 0.02 ± 0.01/s (or 0.3 ± 0.1/s). The direct binding interaction between FVIII and A2 is further confirmed with the surface plasmon resonance assay, with a dissociation constant of ~0.2 μmol/L; no binding is detected between FVIII and A1 under the same conditions. Conclusions: Our results suggest that binding of FVIII to D′D3 and/or A2 may alter the mechanical property in the central A2 domain. The findings provide novel insight into the molecular mechanism underlying FVIII-dependent regulation of VWF proteolysis by ADAMTS13 under mechanical force.