Two novel diorganotin (IV) complexes, based on 4-nitro-N-phthaloyl-glycine (HL), namely {4-NO₂C₆H₃(CO)₂NCH₂COO}₂Sn(n-Bu)₂ (1) and {4-NO₂C₆H₃(CO)₂NCH2COO}₂SnMe₂ (2), were synthesized and characterized by elemental analysis, FT-IR, ¹H- and ¹³C-NMR spectroscopic techniques. In vitro antitumor activities of both complexes were evaluated by the 3-(4,5-dimethylthiazoly-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against three human cancer cell lines: HepG-2 (human liver carcinoma), SGC-7901 (human gastric carcinoma) and LS174T (human colon carcinoma). Complex 1 exhibited strong antitumor activity with IC₅₀ values of 1.51±0.41, 1.80±0.63, and 2.48±0.96μM, respectively; while complex 2 had no obvious effects on the three selected cancer cell lines at high concentrations up to 100μM. Complex 1-induced apoptosis was further confirmed by morphological observations and annexin V-FITC/PI staining flow cytometry analysis in HepG-2 cells. Cell cycle analysis revealed that complex 1 caused cell cycle arrest at G2/M phase. Molecular mechanism studies suggested that the apoptosis was mediated through the mitochondrial pathway with intracellular reactive oxygen species (ROS) promotion and mitochondrial membrane potential (MMP) disruption by finally activating effector caspase-3/9 to trigger cell apoptosis. Moreover, the interactions of both complexes with calf thymus DNA (CT-DNA) were investigated by using UV-Vis titration and fluorometric competition measurements. The DNA-binding constants K_b (intrinsic binding constant) and K_sv (quenching constant) had been obtained in the order: 1>2, consisted with the antitumor activity results. Taken together, complex 1 exhibited excellent antitumor activity suggesting that it may be a potential candidate for further chemical optimization and cancer therapy.