Golgi alpha-mannosidase II (GMII), a member of glycoside hydrolase family 38, cleaves two mannosyl residues from GlcNAcMan(5)GlcNAc(2) 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 GM II. 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 S-o(2)/B-2,B-5 conformation in the GMII Michaelis complex and that the nucleophilic attack occurs before complete departure of the leaving group, consistent with a D(N)A(N) reaction mechanism. The transition state has a clear oxacarbenium ion (OCI) character, with the glycosylation reaction following an S-o(2)/B-2,B-5 -> B-2,B-5 [TS] -> S-1(5) itinerary, agreeing with an earlier proposal based on comparing alpha- and beta-mannanases. The simulations also demonstrate that an active-site Zn ion helps to lengthen the O-2'-H-O2, bond when the substrate acquires OCI character, relieving the electron deficiency of the OCl-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.