In the course of screening plants used in folk medicine as memory enhancers, a 70% ethanolic extract of Stephania rotunda roots showed significant AChE inhibitory activity. Repeated column chromatography led to the isolation of a new protoberberine alkaloid, which we named stepharotudine (1), and seven known compounds (2-8). The chemical structures of the isolated compounds were elucidated based on extensive 1D and 2D NMR spectroscopic data. Compounds 1-8 were investigated in vitro for their anticholinesterase activity using a rat cortex AChE enzyme.