This study is concerned with parametric study of the effect of rectangular web openings on the response of steel bridges with cross frames and bottom lateral bracings. The study includes studying the deflections of steel bridges as an important factor of static response and the frequencies of steel bridges as an important factor of dynamic response. The influence of rectangular web openings of various sizes and numbers on the response of steel bridges is investigated with and without horizontal stiffeners (reinforcements). Buckling and instability failures of the steel beams are not considered in the present analysis. Based on the analysis results of steel bridges with cross frames and bottom lateral bracings, it is shown that existence of only one rectangular (square) web opening at mid span has almost negligible effect on the maximum deflections at mid span and small effect on the frequencies of bridge models. However, existence of more than one square or rectangular web opening leads to great increase in the deflection at mid span and considerable decrease in frequencies. Also, an increase in the ratio of web opening height to web height leads to great increase in the maximum deflection of bridge model. On the other hand, horizontal stiffeners (reinforcements) above and below web openings improves both static and dynamic response of steel bridge models especially, for multiple web openings. However, the effect of reinforcement stiffness is more pronounced for static response.