This study investigated the shear behavior of two reinforced thin-wall box girders with vertical prestressed tendon. Whether the vertical tendons were prestressed or not and the ducts were grouted or not were comparatively studied. The experimental study addressed diagonal cracking behaviors, crack patterns and width, load-deflection relationship, the web strain and stirrup strain, as well as ultimate shear capacity. The test results have shown that, the cracking load of box girder with prestressing force is about 16% higher than those without, and the effect of prestressing force only increases by about 2.3% for ultimate shear capacity. Meanwhile, the shear resistance of girder without grouting is about 9.3% lower than those with ducts well grouted. Through the comparison of test girders, it has been found that the vertical prestressing force can improve the diagonal cracking load and shear capacity. The grouting quality for vertical prestressed ducts is of great importance to the shear behavior of the box girder.