Butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are highly homologous proteins with distinct substrate preferences. In this study we compared the active sites of monomers and tetramers of human BChE and human AChE after performing molecular dynamics (MD) simulations in water-solvated systems. By comparing the conformational dynamics of gating residues of AChE and BChE, we found that the gating mechanisms of the main door of AChE and BChE are responsible for their different substrate specificities. Our simulation of the tetramers of AChE and BChE indicates that both enzymes could have two dysfunctional active sites due to their restricted accessibility to substrates. The further study on catalytic mechanisms of multiple forms of AChE and BChE would benefit from our comparison of the active sites of the monomers and tetramers of both enzymes.