Two new polyynes, named oplopantriol A ( 5) and oplopantriol B ( 6), were isolated from the root bark of Oplopanax horridus (Smith) Miq, an ethnic medicinal plant of North America, along with four known polyynes: (3 S,8 S)-falcarindiol ( 1), oplopandiol ( 2), (11 S,16 S,9 Z)-9,17-octadecadiene-12,14-diyne-1,11,16-triol, 1-acetate ( 3) and oplopandiol acetate ( 4). The structures of the new compounds were elucidated by detailed spectroscopic analyses, including 1D and 2D NMR techniques and chemical methods. The absolute configurations of the new compounds 5 and 6 were determined by comparing their optical rotation values with the hydrolysis products of the known compounds 3 and 4, respectively, derived from the same plant. On the basis of an analysis of their physical and chemical properties we show that the alkaline hydrolysis of 3 and 4 afforded the new compounds 5 and 6, respectively.