In the course of searching for acetylcholinesterase inhibitors from crude drugs, it was found that total MeOH extract of Corydalis Tuber showed significant inhibitory effect on acetylcholinesterase. To isolate acetylcholinesterase inhibitors from Corydalis Tuber, total MeOH extract of the the crude drug was subjected to activity guided fractionation. The MeOH extract was suspended in water and fractionated with methylene chloride and subjected to acid-base fractionation. Silica gel column chromatography of the basic fraction which showed significant inhibitory effect on acetylcholinesterase was carried out and 5 subfractions (1-5) were obtained. From subtraction 4, compound I was isolated. The structure of isolated compound I was identified by spectroscopic parameters of $^1H-NMR$, $^{13}C-NMR$, EI-MS and FAB-MS. The compound I was identified as berberine. It was found from the Lineweaver-Burk plot that berberine was a reversible and specific inhibitor of acetylcholinesterase having 90% inhibitory effect at the concentration of $2.5{mu}M$.