AbstractA deep in silico investigation of various microsomal prostaglandin E₂ synthase‐1 (mPGES‐1) protein systems is here reported using molecular dynamics (MD) simulations. Firstly, eight different proteins models (Models A−H) were built, starting from the active enzyme trimer system (Model A), namely that bound to three glutathione (GSH) cofactor molecules, and then gradually removing the GSHs (Models B−H), simulating each of them for 100 ns in explicit solvent. The analysis of the obtained data disclosed the structural role of GSH in the chemical architecture of mPGES‐1 enzyme, thus suggesting the unlikely displacement of this cofactor, in accordance with experimentally determined protein structures co‐complexed with small molecule inhibitors. Afterwards, Model A was submitted to microsecond‐scale molecular dynamics simulation (total simulation time=10 μs), in order to shed light about the dynamical behaviour of this enzyme at atomic level and to obtain further structural features and protein function information. We confirmed the structural stability of the enzyme machinery, observing a conformational rigidity of the protein, with a backbone RMSD of ∼3 Å along the simulation time, and highlighting the strong active contribution of GSH molecules due to their active role in packing the protein chains through a tight binding at monomer interfaces. Furthermore, the focused analysis on R73 residue disclosed its role in solvent exchange events, probably excluding its function as route for GSH to enter towards the endoplasmic reticulum membrane, in line with the recently reported function of cap domain residues F44‐D66 as gatekeeper for GSH entrance into catalytic site.