Abstract It has been well known that Vertical Central Stabilizers (VCS) have the potential of improving flutter performance of long-span bridges. However, the fundamental flutter mechanisms of VCS are still not fully understood so far. In this study, a series of wind-tunnel tests involving the combination of six representative heights and four types of VCS were conducted to fundamentally investigate the influence of VCS on flutter performance of twin-box girders with various Slot Width Ratios (SWRs). Experimental results show that the flutter instability of 20% SWR is significantly sensitive to the height change of VCS, whereas the VCS have little effect on the flutter performance for 80% and 100% SWR. In addition, the results from Two-Dimensional Three Degree of freedom (2D-3DOF) flutter analysis demonstrates that aerodynamic damping Part A with reference of flutter derivative A 2 ∗ makes the greatest contribution to the flutter instability for a 0.8 h/H VCS, while the role of Part D with reference of A 1 ∗ H 3 ∗ becomes critical for a short VCS (i.e. the ratio of h/H is less than 0.2). Besides, the results of Computational Fluid Dynamics simulation indicate that the geometry of VCS could potentially influence the transforming vortices’ structures and pressure distribution under the central slotting. Finally, the modified Selberg formula presented in this study has the capability of predicting the critical flutter speeds of twin-box girders with various SWRs and VCS.