Abstract An experimental investigation of Reynolds number effects on aerodynamic characteristics and vortex-induced vibration (VIV) of a twin-box girder was performed. The Reynolds number is in the range of 5.85×10 3 ≤Re≤1.12×10 5 when using the central height of the twin-box girder as the characteristic length. The pressure distributions, aerodynamic force and vortex-shedding frequency at various Reynolds numbers were investigated. The results indicate that all the aerodynamic characteristics have obvious Reynolds number sensitivity. According to the mean and fluctuating pressure distributions, the leading separated bubble length on the lower surface was obtained. With increases in Reynolds number, the transition point from laminar flow to turbulence flow and the reattachment point of the separated shear layer gradually move upstream, and the bubble size shrinks. Simultaneously, the drag force coefficient decreases, whereas the Strouhal number increases. In addition, the bubble length has a good linear relationship with the drag force coefficient and with the Strouhal number. The Reynolds number effects on the VIV of a twin-box girder were investigated at three Reynolds number levels: Re=[6.47×10 3 –7.93×10 3 ], [1.17×10 4 –1.62×10 4 ] and [1.87×10 4 –2.90×10 4 ]. The results indicate that the VIV of a twin-box girder has significant Reynolds number dependence. The peak amplitude of the VIV increases with increase in the Reynolds number. The relationship between the peak amplitude of the VIV of the twin-box girder and the Skop–Griffin number at various Reynolds numbers was obtained, which can be employed to predict the peak amplitude of VIV.