Vanadium redox flow batteries (VRFBs) are a promising solution for large-scale energy storage, but a number of problems still impede the deployment of long-lifetime VRFBs. One important aspect of efficient operation of VRFBs is understanding interactions between vanadium species and the membrane. Herein, we investigate the interactions between all four vanadium cations and Nafion membrane by a combination of infrared (IR) spectroscopy and density-functional-theory (DFT)-based static and molecular dynamics simulations. It is observed that vanadium species primarily lead to changes in the IR spectrum of Nafion in the SO 3 − spectral region which is attributed to the interaction between vanadium species and the SO 3 − exchange sites. DFT calculations of vanadium − Nafion complexes in the gas phase show that it is thermodynamically favorable for all vanadium cations to bind to SO 3 − via a contact pair mechanism. Car-Parrinello molecular dynamics-based metadynamics simulations of cation-Nafion systems in aqueous solution suggest that V 2 + and V 3 + species coordinate spontaneously to SO 3 − , which is not the case for VO 2 + and VO 2 + . The interaction behavior of the uncycled membrane determined in this study is used to explain the experimentally observed changes in the vibrational spectra, and is discussed in light of previous results on device-cycled membranes.