Hemoglobin (Hb), an oxygen-binding protein composed of four subunits (α1, α2, β1, and β2), is a well-known example of allosteric proteins that are capable of cooperative ligand binding. Despite decades of studies, the structural basis of its cooperativity remains controversial. In this study, we have integrated coarse-grained (CG) modeling, all-atom simulation, and structural data from X-ray crystallography and wide-angle X-ray scattering (WAXS), aiming to probe dynamic properties of the two structural states of Hb (T and R state) and the transitions between them. First, by analyzing the WAXS data of unliganded and liganded Hb, we have found that the structural ensemble of T or R state is dominated by one crystal structure of Hb with small contributions from other crystal structures of Hb. Second, we have used normal mode analysis to identify two distinct quaternary rotations between the α1β1 and α2β2 dimer, which drive the transitions between T and R state. We have also identified the hot-spot residues whose mutations are predicted to greatly change these quaternary motions. Third, we have generated a CG transition pathway between T and R state, which predicts a clear order of quaternary and tertiary changes involving α and β subunits in Hb. Fourth, we have used the accelerated molecular dynamics to perform an all-atom simulation starting from the T state of Hb, and we have observed a transition toward the R state of Hb. Further analysis of crystal structural data and the all-atom simulation trajectory has corroborated the order of quaternary and tertiary changes predicted by CG modeling. Proteins 2013. © 2012 Wiley Periodicals, Inc.