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: (3S,8S)-falcarindiol (1), oplopandiol (2), (11S,16S,9Z)-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.