The critical role of partially folded intermediates in protein misfolding and amyloid formation has sparked interest in exploring factors that control the formation of these metastable species. Recent NMR experiments reported a sparsely populated intermediate of the villin headpiece domain, in which the N-terminal subdomain is random coil-like under native conditions. Here we use continuous constant pH molecular dynamics simulations with replica-exchange sampling protocol to test the hypothesis that the conformational state obtained by simulations derived from a solution NMR structure represents a putative intermediate in which the N-terminal subdomain is partially unfolded. On the basis of the unique titration behavior of His41 as well as the structural and dynamic properties of this state, we propose that the loss of a hydrogen bond between N delta of His41 and the backbone carbonyl oxygen of E14 in the NMR structure leads to the loss of inter-subdomain contacts as well as partial disruption of the N-terminal hydrophobic cluster. Thus, the loss of the hydrogen-bonded network and not the protonation of His41 is a prerequisite for unfolding.