This study aims to investigate the chemical components of the aerial parts of Chloranthus japonicus and their chemosensitizing activities on human osteosarcoma U2OS cells. Thirteen compounds were isolated and purified from petroleum ether extract of the aerial parts of C. japonicus by column chromatography over silica gel, Toyopearl HW-40C, ODS and semi-preparative high performance liquid chromatography. Their chemical structures were elucidated based on physiochemical properties, spectral datas, ECD calculation and literature comparisons, as chlojaparomatic (1), shizukaol B (2), shizukaol F (3), chlorajaponol (4), shizukaol C (5), shizukaol E (6), sarglabolide I (7), cycloshizukaol A (8), chloranholosin B (9), (–)-loliolide (10), 3-isopropyl-5-acetoxycyclohexene-2-one-1 (11), 2'-hydroxy-4,3',4',6'-tetramethyl chalcone (12) and 5,7,4'-trimethoxyflavanone (13). Among them, compound 1 was one new compound; and 9-13 were isolated from this species for the first time. Bioassay results showed that the dichloromethane extract had an inhibitory effect on human breast cancer MCF-7 cells. The petroleum ether and dichloromethane extracts at 50 and 100 μg/mL had synergistic cytotoxic effects in combination with doxorubicin on U2OS cells. Compound 1 increased the sensitivity of U2OS cells to doxorubicin, and the combination index at 50 μmol/L was 0.77 ± 0.40.