The transcriptional repressor Rex plays key roles in modulating respiratory gene expression. It senses the redox poise of the NAD(H) pool. Rex from Streptomyces rimosus (Sr-Rex) is a newly identified protein. Its structure and complex with substrates are not determined yet. In this study, the three-dimensional (3D) structural models of Sr-Rex dimer and its complex with cofactors were constructed by homology modeling. The stability of the constructed Sr-Rex models and the detailed interactions between Sr-Rex and cofactors were further investigated by molecular dynamics simulations. The results demonstrated that the conformation of Sr-Rex changed a lot when binding with the reduced NADH or oxidized NAD(+). Once binding with NADH, the Sr-Rex dimer displayed an opener conformation, which would weaken the interaction of Sr-Rex with Rex operator DNA (ROP). Key residues responsible for the binding were then identified. The computational results were consistent with experimental results, and hence provided insights into the molecular mechanism of Sr-Rex binding with ROP and NADH/NAD(+), which might be helpful for the development of biosensor.