This study aims to investigate the chemical constituents of diterpenoid alkaloids from the roots of Aconitum pendulum Busch. and their anti-inflammatory activities. The compounds were isolated and purified by using silica gel column chromatography and thin layer chromatography, and the structures of the compounds were identified by spectral analysis and physicochemical properties. Meanwhile, the MTT and Griess methods were used to evaluate the anti-inflammatory activities of compounds. A total of 13 diterpenoid alkaloids compounds were identified as mesaconitine (1), hypaconitine (2), songorine (3), 3-deoxyaconitine (4), aconitine (5), 3-acetylaconitine (6), 2-hydroxydeoxyaconitine (7), 8-O-methylaconitine (8), 3-deoxyaconine-8-linoleate (9), 14-benzoylaconine-8-oleate (10), 14-benzoylaconine-8-palmitate (11), 14-benzoylaconine-8-linoleate (12), azitine (13). Compounds 9, 10-13 were isolated from the roots of A. pendulum for the first time. Compounds 1-6, 9-12 all showed inhibition effects on NO production in RAW 264.7 cells and have certain anti-inflammatory activity. ELISA assays revealed that compounds 4, 5, 9-12 could reduce the expression of inflammatory cytokines interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α). Compound 12 has a relatively strong anti-inflammatory effect, suggesting its potential value for further in-depth research.