Abstract Human butyrylcholinesterase (BChE) and its mutants have shown great potential in treating cocaine overdose and addiction. In order to effectively suppress cocaine reward in the brain for a long period of time after an exogenous cocaine hydrolase administration, the therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long circulation time. It has been known that PEGylation (covalent attachment of polyethylene glycol) modification of a therapeutic protein can prolong the biological half-life of the protein without affecting its biological function. However, the asparagine-linked glycans on the surface of glycosylated BChE may interfere with the PEGylation modification. In this study, we built a three-dimensional (3D) model of glycosylated human BChE to investigate the influence of glycans on the PEGylation modification. Glycans did not change the overall stability of the BChE structure, but could increase the flexibility of some local structures. For further evaluating the accessibility of the PEGylation reaction sites, particularly lysine residues, on the protein surface, we calculated the Solvent Accessible Surface Areas (SASAs) of these residues. The results indicate that some lysine residues show a significant decrease in SASA due to the direct or indirect influence of their surrounding glycans. The results also indicate that PEGylation reaction agents with smaller functional groups could have a better chance to react with lysine residues. This investigation provides a structural basis for rational engineering of human BChE and its mutants as therapeutic candidates.