Objective To reveal the shear stress regulation mechanism on vWF-A1A2A3-mediated rolling of platelets in flows. Methods Platelets were perfused with various flow rates over 200 μg/mL vWF A1A2A3 in parallel-plate flow chamber. The rolling events of platelets near the bottom of chamber were observed and analyzed to obtain the rolling characteristics. Results The mean rolling velocity and time, stop frequency and fractional stop time of platelets were all found to be biphasic dependent on wall shear stress, with all the threshold/inflection points occurring under the fluid stress of 0.8 Pa. Increasing shear stress below the threshold would slow and stabilize cell rolling in flows. Conclusions Wall shear stress regulates velocity of A1A2A3-mediated cell rolling by adjusting the cell stop time, suggesting that a catch-to slip-bond transition serves as a mechanical regulation mechanism for platelet rolling through A1A2A3 GPIbα interaction in flows.