Prolyl oligopeptidase (POP) is a serine protease, unique for its ability to cleave various small oligopeptides shorter than 30 amino acids. POP is an important drug target since it is implicated in various neurological disorders. Although there is structural evidence that bacterial POPs undergo huge interdomain movements and acquire an “open” state in the substrate-unbound form, hitherto, no crystal structure is available in the substrate-unbound domain-open form of eukaryotic POPs. Indeed, there is no difference between the substrate-unbound/bound states of eukaryotic POPs. This raises unanswered questions about whether difference in the substrate access pathway exists between bacterial and eukaryotic POPs. Here, we have used normal mode analysis and molecular dynamics to unravel the mechanism of substrate entry in mammalian POPs, which has been debated until now. Motions observed using normal modes of porcine and bacterial POPs were analyzed and compared, augmented by molecular dynamics of these proteins. Identical to bacterial POPs, interdomain opening was found to be the possible pathway for the substrate-gating in mammals as well. On the basis of our analyses and evidences, a mechanistic model of substrate entry in POPs has been proposed. Up-down movement of N-terminal hydrolase domain resulted in twisting motion of two domains, followed by the conformational changes of interdomain loop regions, which facilitate interdomain opening. Similar to bacterial POPs, an open form of porcine POP is also proposed with domain-closing motion. This work has direct implications for the development of novel inhibitors of mammalian POPs to understand the etiology of various neurological diseases. Proteins 2014; 82:1428–1443. © 2014 Wiley Periodicals, Inc.