An enantiomeric pair of rare cyperane-type sesquiterpenoids, (+)- and (-)-gracilistones C (<b>1a</b>, <b>1b</b>), together with a novel norsesquiterpenoid, gracilistone D (<b>2</b>), bearing a bicyclic lactone system were isolated from the root bark of <i>Acanthopanax gracilistylus</i> using LC-MS-IT-TOF analyses. The structures and absolute configurations of <b>1a</b>, <b>1b</b>, and <b>2</b> were elucidated by 1D and 2D NMR spectroscopy, X-ray diffraction, and ECD spectroscopic methods. Intermediate <b>1b</b> suggests a possible biosynthesis process involving compound <b>2</b>. The bioassay results showed that compounds <b>1a</b>, <b>1b</b>, and <b>2</b> exhibited significant inhibitory effects against lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells, with IC<sub>50</sub> values of 7.7 ± 0.6, 6.8 ± 1.5, and 2.6 ± 0.4 μM, respectively. Additional docking analyses provided some perspective of this activity in human inducible nitric oxide synthase.