This study aims to research the chemical constituents of the petroleum ether extract from Tripterospermum filicaule (Hemsl.) Harry Sm (Gentianceae). The chemical compositions were isolated and purified by Sephadex LH-20 gel and silica gel column chromatography. The chemical structures of isolated compounds were elucidated by NMR and MS methods. The cytotoxic activity on human liver cancer HepG2 cells of the chemical constituents from the petroleum ether extract of T. filicaule was detected using the CCK-8 method . Twenty-two compounds were isolated and identified from the petroleum ether fraction of the herb of T. filicaule. These compounds were identified as balanophorin B (1), 3β-palmitate-28-hydroxyl-β-amyrin (2), 3β-palmitate-28-hydroxyl- α-amyrin (3), uvaol (4), 2-hydroxydiploptero (5), eupatoric acid (6), oleanolic acid (7), ursolic acid (8), stigmasta-4-en-3-one (9), stigmasta-4,22-dien-3-one (10), stigmasterol (11), β-sitosterol (12), (22E)-5a,8a-epidoxyergosta-6,22-dien-3b-ol (13), 1,8-dihydroxy-3,4,7-trimethoxyxanthone (14), physcion (15), α-tocopherolquinone (16), 1,8-dihydroxy-3,7-dime-thoxyxanthone (17), pentadecanoic acid (18), 1,7-dihydroxy-3,4-dimethoxyethanone (19), hentriacontanol (20), 2,2'-oxybis(1,4-di-tert-butylbenzene) (21), and trilinolein (22). Compounds 1-6, 9, 10, 13-22 were isolated from the Tripterospermum species for the first time, all the compounds were first isolated from T. filicaule. The activity screening results showed that 7, 8, and 15 had certain inhibitory effects on liver cancer HepG2 cells, with IC₅₀ values of 56.72 ±0.65, 49.93 ± 0.93, and 47.65 ± 0.58 μmol/L, respectively.