Four xanthones were isolated from Gentianopsis paludosa Ma and were identified by modern spectroscopic methods. Cytotoxicity of the four xanthones was tested on HepG2 cells and HL-60 cells by sulphorhodamine B (SRB) assay. Clonogenic survival assay, trypan blue exclusion method, AO/EB staining and DNA fragmentation assay were conducted to investigate the effect on growth inhibition and apoptosis in the two cell lines in vitro. At the same time, structure-activity relationships (SARs) of the xanthones were investigated. The results showed that the xanthones had significant cytotoxicity and inhibition of proliferation in both HepG2 cells and HL-60 cells, and could induce apoptosis in these two cell lines. SARs indicated that the methoxy group had more cytotoxic contribution than the hydroxyl group at site C-8 in the structural scaffold of xanthone. The glycosidea at site C-1 may aggravate the stereospecific blockade of compound 4 and reduced its cytotoxic activity.