Prolyl oligopeptidase (POP) has gained importance as a target for the treatment of neuropsychiatric diseases and cognitive disturbances. Therefore, a variety of strategies are currently used to identify POP inhibitors. Here we performed electron microscopy (EM) studies of human POP. Our data reveal for the first time the presence of a new side opening in POP that was not observed in any of the crystallographic structures described to date. Finally, molecular dynamics, the relevant normal modes that contribute to the fluctuation of the catalytic triad residues and the algorithm CAVERN also support the existence of a new large side opening on POP.