Golgi α-mannosidase II (GMII), a member of glycoside hydrolase family 38, cleaves two mannosyl residues from GlcNAcMan5GlcNAc2 as part of the N-linked glycosylation pathway. To elucidate the molecular and electronic details of the reaction mechanism, in particular the conformation of the substrate at the transition state, we performed quantum mechanics/molecular mechanics metadynamics simulations of the glycosylation reaction catalyzed by GMII. The calculated free energy of activation for mannosyl glycosylation (23 kcal/mol) agrees very well with experiments, as does the conformation of the glycon mannosyl ring in the product of the glycosylation reaction (the covalent intermediate). In addition, we provide insight into the electronic aspects of the molecular mechanism that were not previously available. We show that the substrate adopts an OS2/B2,5 conformation in the GMII Michaelis complex and that the nucleophilic attack occurs before complete departure of the leaving group, consistent with a DNAN reaction mechanism. The transition state has a clear oxacarbenium ion (OCI) character, with the glycosylation reaction following an OS2/B2,5 → B2,5 [TS] → 1S5 itinerary, agreeing with an earlier proposal based on comparing α- and β-mannanases. The simulations also demonstrate that an active-site Zn ion helps to lengthen the O2′−HO2′ bond when the substrate acquires OCI character, relieving the electron deficiency of the OCI-like species. Our results can be used to explain the potency of recently formulated GMII anticancer inhibitors, and they are potentially relevant in deriving new inhibitors.