This study aims to invstigate the chemical constituents from Pinelliae Rhizoma and their anti-inflammatory activity.Twenty-six compounds were isolated and purified from the ethyl acetate extract of Pinelliae Rhizoma silica gel,MCI,ODS and Toyopearl HW-40C column chromatography and semi-preparative HPLC methods.Their chemical structures were elucidated by physiochemical properties and spectral data as cyclo-(L-Pro-8-hydroxy-D-Ile) (1),cyclo-(L-Pro-L-Leu) (2),cyclo-(L-Pro-L-Phe) (3),cyclo-(L-Pro-L-Val) (4),adenosine (5),5′-S-methyl-5′-thioadenosine (6),2′-methoxyadenosine (7),adenine (8),1,8,15-triazecycloterpine-2,9,16-trione (9),1,8,15,22-tetraazacyclooctacosane-2,9,16,23-tetraketone (10),1,8,15,22,29-pentaaza-pentadecane-2,9,16,23,30-pentanone (11),diethylene glycol azelate (12),glycerol monopalmitate (13),glycerin monolinoleate (14),9,12-octadecadienoic acid N-(2-hydroxyethyl) (15),2-butoxyethyl linolenate (16),(2S)-1-O-(9Z,12Z-octadecandienyl)-3-O-β-galactosyl glycerol (17),β-sitosteryl-3β-glucopyranoside-6′-O-palmitate (18),bis (2-ethylhexyl) phthalate (19),lariciresinol (20),lariciresinol-9-O-β-glucopyranoside (21),(+)-epipinoresinol-4′-O-β-glucopyranoside (22),(+)-isolariciresinol-9′-O-β-glucopyranoside (23),(+)-isolariciresinol-9-O-β-glucopyranoside (24),vanillic acid (25),and (E)-p-coumaroyl alcohol (26).Compounds 1,3,4,8-12,15,16,19 and 23 were isolated from Pinelliae Rhizoma for the first time. Some of isolated compounds were further evaluated for their anti-inflammatory activity in lipopolysaccharide (LPS)-induced mouse macrophage (RAW 264.7) cells. The results showed that compounds 2,3,11,16 and 21 had inhibitory effects on NO release, and compound 16 had stronger NO inhibition effect with IC₅₀ values of 10.47 ± 0.89 μmol/L.