In this paper, the moment of inertia requirement of flat-bar longitudinal stiffener of bottom flange in steel box girder is investigated through finite element modeling. The required minimum stiffness for longitudinal stiffeners of box girder flange is given in the AASHTO LRFD Bridge Design Specification and is modified by Yoo. However, this requirement is adopted for T-shape stiffeners. Here, the effect of important parameters on the minimum required moment of inertia of flat-bar stiffener is numerically investigated by examining the anti-symmetric mode of buckling. This study presents the results that are based on 3D finite-element analysis of four hundred hypothetical compression flange models stiffened by varying numbers of flat-bar longitudinal stiffeners with realistic dimensions such as the height of stiffener, the thickness of the compression flange, the number of longitudinal stiffeners and the aspect ratio of plate panel. A new proposed equation for required minimum stiffness of the flat-bar longitudinal stiffeners is derived from nonlinear regression analyses. Beside that the study has taken into account the effect of boundary conditions and the effect of inelastic transition on the critical buckling stress of compression flange. Through the evaluation of a design example, the validity and reliability of the new proposed equation is demonstrated.