Lots of fatigue crackings were reported at the upper end of vertical stiffeners connected to sway bracings in steel highway girder bridges. In this study, we investigate fatigue cracking behavior at the upper end of vertical stiffeners under RC slab, through fatigue tests using a large specimen with three main girders under alternative loading using two actuators. As a result, alternative loading can reproduce the alternative stress at the upper end of vertical stiffener in the middle girder when vehicles run on the driving lane and passing lane alternatively. Root cracks were initiated after 0.1 Mcycles loading and appeared on the bead surface when 0.6 Mcycles.