The ethanol extract of Punica granatum L. rind was tested to show significant nematicidal activity against pine wood nematode. Three nematicidal compounds were obtained from the ethanol extract by bioassay-guided fractionation and identified as punicalagin 1, punicalin 2, and corilagin 3 by mass and nuclear magnetic resonance spectral data analysis. Punicalagin 1 was most active against PWN among the purified compounds with the LC50 value of 307.08μM in 72h. According to the enzyme assays in vitro, punicalagin 1 could inhibit the activity of acetylcholinesterase, amylase and cellulase from PWN with IC50 value of 0.60mM, 0.96mM and 1.24mM, respectively. The morphological structures of PWNs treated by punicalagin 1 were greatly changed. These physiological effects of punicalagin 1 on PWN may helpful to elucidate its nematicidal mechanism.