The misfolding of the prion protein has been linked to several neurodegenerative diseases. Despite extensive studies, the mechanism of the misfolding process remains poorly understood. The present study structurally delineates the role of the conserved proline residues present in the structured C-terminal domain of the mouse prion protein (moPrP) in the misfolding process. It is shown that mutation of these Pro residues to Ala leads to destabilization of the native (N) state, and also to rapid misfolding. Using hydrogen-deuterium exchange (HDX) studies coupled with mass spectrometry (MS), it has been shown that the N state of moPrP is in rapid equilibrium with a partially unfolded form (PUF2*) at pH 4. It has been shown that the Pro to Ala mutations make PUF2* energetically more accessible from the N state by stabilizing it relative to the unfolded (U) state. The apparent rate constant of misfolding is found to be linearly proportional to the extent to which PUF2* is populated in equilibrium with the N state, strongly indicating that misfolding commences from PUF2*. It has also been shown that the Pro residues restrict the boundary of the structural core of the misfolded oligomers. Overall, this study highlights how the conserved proline residues control misfolding of the prion protein by modulating the stability of the partially unfolded form from which misfolding commences.