To account for protein dynamics and to investigate the effect of different conformations on the enzyme reaction energy barrier, we have studied the initial step of the acylation reaction catalyzed by acetylcholinesterase (AChE) with a multiple QM/MM reaction path approach. The approach consists of two main components: generating enzyme-substrate conformations with classical molecular dynamics simulation and mapping out the minimum reaction energy path for each conformational snapshot with combined quantum mechanical/molecular mechanical (QM/MM) calculations. It is found that enzyme-substrate conformation fluctuations lead to significant differences in the calculated reaction energy barrier; however, the qualitative picture of the role of the catalytic triad and oxyanion hole in AChE catalysis is very consistent. Our results emphasize the importance of employing multiple starting structures in the QM/MM study of enzyme reactions and indicate that structural fluctuation is an integral part of the enzyme reaction process.