In 2017, Specifications for highway bridges in Japan were revised from the allowable stress design method into the partial factor design method. The new methods and the partial factors for each limit state should be proposed according to provisions described in the revised specifications for highway bridges. In this study, the four-point bending tests on two test specimens with a horizontal stiffener were conducted in order to establish the more rational design method of steel girders by considering the behavior in the plastic region and the increasing the width-thickness ratio of web panels. Based on the experimental results, the bending strength of steel girders, the relationship between the load and displacement, the out-of-plane deformation and the strain distributions were investigated. Furthermore, the applicability of the plastic design method to steel girders and the possibility of the increase in the width-thickness ratio of web panels were examined.